Neurotrophins in developing human inner ear and in HNSCC

Project Focus The neurotrophins are growth factors that play important roles in the development of human inner ear. These factors are also involved in failure of standard chemotherapy in head and neck cancer. These two important areas of otorhinolaryngology will cooperate to find mechanisms involved in hearing loss on one side and tumour therapy failure on the other. The key word is survival. Neurotrophins support the survival of functional neurons in inner ear as well as the survival of tumour cells after radio / chemotherapy. Hypothesis Neurothophins show a gradient during inner ear development, which define developmental patterns by induction of various genes. Neurotrophins contribute to tumour cell survival in radio / chemotherapy treated patients. Methods: In this project the most modern methods as gene expression profiling, laser based cutting out of cells from inner ear and tumour tissue and the newest microscopical methods will be utilized. The Innsbruck ENT Clinic will provide an array of inner ear samples in different stages of inner ear organ formation and induction of the hearing function, while the Matsumoto Group will identify the presence of genes that get wrong by hearing loss diseases. This project will define functions for genes that prevent the success of cochlea implants. In head and neck cancer, labor bench work will be extended by analysis of human tissue samples in order to understand the mechanism of neurotrophin related therapy failure. What is new in this project? Two important outcomes are new in this project: 1. The genes involved in hearing loss will be investigated in inner ear development and it will be found out how these genes are involved in what goes wrong by hearing loss. 2. The neurotrophins are involved in therapy failures by head and neck cancer. Identification of the reasons of this failure will help to achieve improved tumor therapy.

Project Focus Hearing loss is one of the most common sensory disorders worldwide, caused by over 120 gene defects. In a cooperation between the Shinshu University in Matsumoto, Japan and the Medical University of Innsbruck, Austria we performed gene expression profiling in developing human inner ear. Neurotrophins are growth factors, which are essential in inner ear development. In our project we focused on brain-derived neurotrophic factor (BDNF) and its receptors. Its cell survival supporting and guidance functions suggested that BDNF might be also involved in the invasion and therapy survival of head and neck squamous cell carcinoma (HNSCC) tumor cells. In HNSCC the tumor stroma provides sufficient amounts of neurotrophins, the main regulation exposes the receptors. Main Methods Normal fetal human inner ears from gestational weeks (GW) 10-19 were used. High throughput next generation sequencing was complemented with in situ hybridization (ISH) and immunohistochemistry (IHC) on histological sections. In HNSCC we investigated the gene expression conditions of BDNF receptor TrkB by polymerase chain reaction (PCR) and by IHC. TrkB and downstream signal pathways were experimentally challenged by commercial inhibitors. Main Results Our studies revealed that spiral ganglion neurons and the sensory epithelium produce peaked levels of BDNF in GW10, and again in GW15-17 in the spiral ganglion and in GW15-16 in the sensory epithelium that correlates with important developmental steps. Several hearing loss causative genes also show expression peaks at GW15-16. The main cells producing BDNF are spiral ganglion neurons, but even more satellite glia cells. The mission of neurotrophins in the inner ear include nerve guidance, maturation and trophic effects. Certain types of neurotrophins complement each other during the formation of our hearing organ and show similarities and differences to other mammals. Human papilloma virus (HPV)-positive HNSCC tissue had higher expression of TrkB compared to HPV-negative tissue. Immunohistochemical and experimental data confirmed the relation between transforming-growth-factor-beta (TGF-beta) and BDNF/TrkB function. Pharmacological inhibition of signal pathways downstream from TGF-beta stimulation and neurotrophin receptors activation, made resistant tumor cells responsive to Cisplatin. What is the main success in this project? We generated three differential gene expression databases of human fetal inner ears with next generation sequencing. Neurotrophins, neurotrophin receptors and hearing loss causative gene expression interplay in a timely regulated process. Our highly specific mRNA and protein signal readouts allowed mRNA quantification and subcellular protein localization to provide us new insights into the precisely orchestrated coordination of cell migration and differentiation to build the complex inner ear architecture. In HNSCC we developed therapy resistance models, where the function of signal pathway inhibitors can be attributed to modification of therapy response.