Potential Role of the Nervous System in Tumorigenesis. A Translational Study of Gastric Cancer
The goal of this project was to answer the question of “Will denervation surgically and/or pharmacogically suppresses the cancer progression not only at the early stage but also at the late stage of gastric cancer in the mice and patients?”. Part I: Denervation suppresses gastric tumorigenesis (published in Scinene Translational Medicine 2014): Surgical denervation via vagotomy during the preneoplastic stage of tumorigenesis diminished tumor incidence and size, and attenuated tumor cell proliferation specifically in the denervated portion of the stomach, suggesting that the vagus nerve promotes gastric cancer growth. Consistent with this idea, pharmacologic denervation via local administration of botulinum toxin type A (BTxA) in the gastric wall similarly impaired preneoplastic growth. Furthermore, surgical or pharmacologic denervation at later stages of tumorigenesis suppressed gastric cancer progression and augmented the antitumor effect of chemotherapy in tumor-bearing mice, resulting in prolonged survival. This effect was mediated in part by inhibition of WNT signaling. In patients with gastric cancer, WNT signaling was associated with neural pathways, and neuronal density was correlated with more advanced tumors. These findings identify nerves as important regulators of gastric stem cell expansion and tumor progression, and suggest M3R as a potential therapeutic target in gastric cancer. Part II: Endoscopic injection of Botulinum toxin A for treatment of gastric cancer (To be published): A pilot clinical study and further in vitro and animal testing: In this project, we have performed a clinical trial (https://clinicaltrials.gov/ct2/show/NCT01822210) to further evaluate the safety and feasibility of endoscopic injection of BTxA into gastric tumor. Patient criteria were gastric adenocarcinomas with locally non-resectable and/or with distant metastasis and lack of response or non-tolerance to 2nd line chemotherapy. BTxA injection directly into and around the tumor was given at dose of 100 U. There were no complications and no adverse effects during or up to 20 weeks after the procedure. Four out six patients survived for an average of 11.5 weeks. One patient was followed up until 20 weeks showing tumor growth but reduced serum level of neuron-specific enolase during the first 8 weeks and no tumor growth afterwards. A human gastric cancer cell line and a mouse model of gastric cancer were also used to test combined treatment of BTxA and chemotherapy. BTxA directly inhibited cell proliferation by 10% and showed synergistic effects with Oxaliplatin in vitro, and even more so in vivo, with marked reduction in tumor size and pathological score. This clinical study demonstrated the safety and feasibility of BTxA injection into the tumor as a potential treatment for gastric cancer. Part III: Nerve growth factor promotes gastric tumorigenesis through aberrant cholinergic signaling (published in Cancer Cell on Dec. 15, 2016): Within the gastrointestinal stem cell niche, nerves help to regulate both normal and neoplastic stem cell dynamics. We have further revealed the mechanisms underlying the cancer-nerve partnership. We found that Dclk1+tuft cells and nerves were the main sources of acetylcholine (ACh) within the gastric mucosa. Cholinergic stimulation of the gastric epithelium induced nerve growth factor (NGF) expression, and in turn conditional overexpression of NGF within gastric epithelium expanded enteric nerves and promoted carcinogenesis. Ablation of ACh-producing Dclk1+cells, blockade of NGF/Trk signaling or vagotomy inhibited epithelial proliferation and tumor development in a muscarinic acetylcholine receptor-3 (M3R)-dependent manner, in part through suppression of Hippo signaling pathway and Yes-Associated Protein (YAP) function. This feed-forward ACh-NGF axis activates the gastric stem cell niche in cancer and offers a compelling target for tumor treatment and prevention.
Conventional cancer therapies such as chemotherapy and radiotherapy that non-selectively inhibit cell division are often associated with severe adverse effects. In this project, we have demonstrated that surgical (vagotomy) or pharmacological denervation of the stomach (with local injection of BTxA) reduced tumour incidence and progression in mouse models of gastric cancer. Vagotomy or BTxA treatment also enhanced the therapeutic effects of systemic chemotherapy suggesting additive or synergistic effects with conventional chemotherapy. Pharmacological inhibition or genetic knockout of the muscarinic acetylcholine M3 receptor suppressed gastric tumorigenesis. The underlying mechanism was involved with WNT and Hippo signalling pathways, stem cell expansion, and nerve growth factor. In human gastric cancer patients, we found that tumour stage correlated with neural density and activated Wnt signalling, whereas vagotomy reduced the risk of gastric cancer. Furthermore, we have recently completed a clinical phase I trial on patients with advanced gastric cancer (https://clinicaltrials.gov/ct2/show/NCT01822210). The results show that endoscopic injection of BTxA into the tumour was safe and might be considered as a potential treatment of gastric cancer.
Intragastric Injection of Botulinum Toxin A to Treat Gastric Cancer: An Open-Label Phase II Clinical Trial
Blocking vagal nerve signals with Botulinum toxin A (Botox) injection into the tumor area suppresses tumor growth in mouse models of gastric cancer.The results of this clinical trial in six patients demonstrate the feasibility and the safety of this procedure as a potential treatment for advanced gastric cancer.
Background/aims: Previously, we demonstrated that blocking vagal nerve signals surgically (by vagotomy) or with Botulinum toxin A (Botox) injection suppresses tumor growth in mice. In this study, we evaluated the safety and possible efficacy of endoscopic therapy with Botox for patients with advanced gastric cancer, and furthermore investigated a combination therapy of Botox plus oxaliplatin in cell culture and in an animal model of gastric cancer. Methods: Patients eligible for this study included those (ECOG 0-2) with verified gastric adenocarcinomas that were locally non-resectable and/or with distant metastasis. The criteria also included a lack of response or non-tolerance to second line chemotherapy. Endoscopic Botox injection was performed under sedation with midazolam. One hundred units Botox were diluted into 14 ml saline. Patients received a total of 7 injections with 2 ml/14.3 units of Botox solution; 3 injections were made directly into the tumor and 4 injections around the tumor. The safety evaluation was performed based on the Common Toxicity Criteria. The study was conducted in accordance with the guidelines for Good Clinical Practice, and was approved by the Regional Committee for Medical and Health Research Ethics and the Norwegian Medicines Agency. A human gastric cancer cell line MKN74 and a well-established genetically engineered mouse model of gastric cancer (INS-GAS FVB mice) were used to test the combination therapy. Results: During the time period from Aug. 2014 - Oct. 2015, six patients were treated with endoscopic injection of Botox. There were no complications in connection with the procedure and no adverse effects during or up to 20 weeks after the procedure. Four (4) out 6 patients survived for an average of 11.5 weeks after Botox treatment. One patient has been followed up for 20 weeks, with an absence of tumor growth between 8 to 20 weeks, while the last patient has significantly improved clinical condition and will have the 8-week postoperative CT scan on November 25, 2015. Botox directly inhibited cell proliferation in vitro by 10% and had an additive effect on oxaliplatin-induced inhibition. In INS-GAS mice with advanced gastric cancer (12 months of age, male:femal=1:1), local injections of Botox (once a month) had a synergistic effect with oxaliplatin treatment (given by i.p. injections for 2 months) in terms of pathological criteria (dysplasia and gastric histologic activity index) and tumor size. Conclusions: The preliminary results of this clinical trial demonstrate the feasibility and the safety of this procedure as a potential treatment for advanced gastric cancer. To further demonstrate the efficacy of Botox endoscopic treatment in combination with chemotherapy, a randomized, double-blind, and placebo-controlled trial with an open-label extension study is in preparation.
A Translational Study of Gastric Cancer (continuation)
Prolongation of the project is needed in order to complete the clinical trial by enrolling 10 patients and refine preclinical and clinical trials to circumvent the difficulties in enrolling patients.
Initial plan and updated plan for 1-year prolongation: 1. Preclinical trial of vagotomy: In this experiment, we plan to determine whether vagotomy will retard cancer in mice with early or advanced dysplasia. A group of 50-100 INS-GAS mice will be subjected to unilateral (anterior) vagotomy at 8, 10 and 12 months of age, time points at which dysplasia has been initiated in this animal model. This has been completed in time. 2. Preclinical trial of botox: In this experiment, a group of 50-100 INS-GAS mice at 8, 10 and 12 months of age will be subjected to botox treatment. Experimental groups will include botox in anterior and posterior sides of stomach (vs. saline control group), botox in anterior side of stomach (vs. posterior side), and botox plus vagotomy (anterior side and two sides). This has been completed in time. 3. Novel target CD44: In this experiment, we will test this hypothesis by analyzing Wnt pathway in details in above experiments. Immunohistochemistry, Western blot, and qRT-PCR analyses of CD44 and ß-catenin will be performed. This has been completed in time. We plan to treat the INS-GAS mice with compounds that have been tested in other animal models to target CD44. This has been modified because we found new evidence of acetylcholine-depended but Wnt ligand-independent Wnt/ß-catenin signalling in leucine-rich repeated containing G protein-coupled receptor 5-positive stem cells. Accordingly, we have treated the mice with M3 receptor antagonist. 4. The cross-talk between tumorigenesis and neurogenesis: In this experiment, we plan to examine whether the neurogenesis and tumorigenesis develop in parallel, independent, or not develop at all in the area that tumor has been removed by acetic acid. If the tumor develops, we will further examine the effects of unilateral, bilateral vagotomy with or without botox treatment. This has been completed in time. 5. Clinical trial of vagotomy/botox: If results from above mechanism-based preclinical trials show detectable therapeutic benefits, a clinical trial will be started. Clinical outcomes, either tumor shrinkage, increased progression-free-survival and overall survival or relapses and failure, will be translated further for clinical use or back into refined preclinical trials aiming to understand and circumvent the limitation. The clinical trial of botox for gastric cancer has been started at St. Olavs Hospital. Two patients have been treated and followed up, and one patient is under evaluation for enrolling the trial. Prolongation of the project is needed in order to complete the clinical trial by enrolling 10 patients and refine preclinical and clinical trials to circumvent the difficulties in enrolling patients.
Potential Role of the Nervous System in Tumorigenesis. A Translational Study of Gastric Cancer
The research hypothesis is that the nerve plays an important role in creating the tumor microenvironment that contributes to tumorigenesis. Therefore, our research question is “Will denervation surgically and/or pharmacologically suppresses the cancer progression not only at the early stage but also at the late stage of gastric cancer in the mice and patients?”.
To answer this question, we have performed the following five studies:
1. Preclinical trial of vagotomy: 129 gastric cancer mice at either the early state (at 8 months of age) or the late stage (at 18 months) have been included. We found that vagotomy reduced the tumour size and the degree of dysplasia. The survival rate was improved from 48% to 71% when vagotomy was performed at the early stage. Furthermore, by genome-wide gene expression profiling, qRT-PCR arrays and immunohistochemistry, we found a relatively specific and long-lasting effect of vagotomy on the Wnt signaling. These results were presented in international conferences, and are included in a manuscript that is pending publication in Science Translational Medicine.
2. Preclinical trial of botox: 268 gastric cancer mice have been included. We found that Botox treatment reduced tumour size, cell proliferation rate, scores of dysplasia and metaplasia. The combination of either Botox or vagotomy with chemotherapy led to a significant increase in survival compared to chemotherapy alone. Furthermore, in vitro stem cell experiments show that the effect of Botox is likely mediated by acetylcholine-mediated Wnt signaling in gastric stem cells. These results have been submitted for presentation at international conference (e.g. DDW2014), and are included together with the vagotomy trial in the manuscript that is pending publication in Science Translational Medicine.
3. Novel target CD44: CD44 is a target of the Wnt pathway. Initially, we planned to treat the gastric cancer mice with compounds targeting CD44, e.g. salinomycin. Based on our new results, we have examined N-Methyl-N-nitrosourea (MNU)-induced gastric cancer in acetylcholine M3 receptor gene knockout mice vs. wild-type controls, and found that M3 receptor knockout mice had fewer tumour incidence and smaller tumour size. Furthermore, we have treated the gastric cancer mice with M3 receptor antagonist darifenacin with or without chemotherapy, which is also suggested by the reviewers of Science Translational Medicine. The animal experiment will be completed in March 2014. Pathological and biological analyses will be completed before the summer 2014.
4. The cross-talk between tumorigenesis and neurogenesis: By application of acetic acid topically to the serosal side of the stomach to induce a prompt necrosis of tumour in the gastric cancer mice, we found that vagotomy delayed tumour regeneration. Furthermore, we have examined effects of acetic acid on human and rat gastric cancer cells as well as the normal gastric mucosal cells in vitro, and found that acetic acid killed human and rat cancer cells more powerfully than normal gastric mucosal cells by cytotoxicity rather than apoptosis. The manuscript has been accepted for publication in J Gastroenterol Hepatol 2014.
5. Clinical trial of vagotomy/botox: Based on these studies, we have initiated two clinical studies in collaboration with St. Olav’s Hospital (Gøran Andersen and Jon Grønbech): 1) Innervasjon i magesekken og prognose ved cancer ventriculi, and 2) Botox® og kreft i magesekken. Both protocols have been approved by REK. In the first trial, 17 gastric cancer patients have been included, and the surgical specimens have been collected. Pathological and immunohistochemical analyses will be completed in April. Microarray and metabolomics analyses will be conducted during a period from February to August 2014. In the 2nd trial, there are no suitable patients yet at St. Olavs Hospital. Therefore, we have shared our protocol with the Rigshospitalet in Copenhagen (Dr. Lars Bo Svendsen) hoping to enroll their patients.
Potential Role of the Nervous System in Gastric Tumorigenesis
Gastric cancer remains the 2nd leading cause of cancer mortality worldwide, and 5-year survival rate is less than 20%-25%. In this translational project, we are studying the molecular mechanism of gastric tumorigenesis in general and the role of the vagal nerve in the tumorigenesis in particular in both animal models and patients.
According to the plan of the project, we have made the significant progress as shown by the following:
1. Identify a possible novel target strategy for gastric anticancer therapy, i.e., ATG-5-ATG-12-ATG-16 complex formation of autophagy. In our two original articles, we have shown evidence for an impairment of autophagy, particularly in this later stage of the pathway, in the tumorigenesis of gastric carcinoids as well as adenocarcinomas in both rodent models and patients. Based on these findings and literature, we have been invited to contribute to a book chapter in Frontiers of Gastrointestinal Research (Karger). These studies have been performed in collaboration with St. Olav’s Hospital, Columbia University and Yale University.
2. Demonstrate that denervation suppresses gastric tumorigenesis. In this study, we have completed the animal experiments and found further evidence that Botox treatment synergizes the effects of systemic chemotherapy (5-FU + oxaliplatin). We have also performed analysis of gene expression profile and protein expression of stem and neural markers in two cohorts of gastric cancer patients and a retrospective cohort study of patients with gastric stump cancer. Taken our original findings (NTNU and St. Olav’s Hospital) together with findings by Columbia University, MIT, and Technology University of Munich, National Cancer Center Hospital of Japan, we are preparing a manuscript for publication.
3. Clinical studies of gastric cancer. Based on these studies, we have initiated two clinical studies in collaboration with St. Olav’s Hospital (Gøran Andersen and Jon Grønbech): 1) Innervasjon i magesekken og prognose ved cancer ventriculi, and 2) Botox® og kreft i magesekken. Both protocols have been approved by REK.
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