Insight into the role of GDNF and the GDNF family of receptors in glioblastoma

Glioblastoma is the most aggressive and the most common type of primary brain cancer, and despite treatment consisting of surgical tumor removal, radiotherapy and chemotherapy, glioblastoma patients have a poor prognosis with a median survival of 15 months and a five-year survival of 5-7 %. Identifying novel prognostic factors and therapeutic targets are of utmost importance in order to identify the fraction of patients with longest survival and in order to improve the treatment response. The glial cell line-derived neurotrophic factor (GDNF) is an important neuronal growth factor, which plays an essential role in neuronal development and maintenance. Four GDNF family receptors have been identified, namely GDNF family receptor alpha 1-4 (GFRA1-4). The rearranged during transfection (RET) receptor is the primary downstream co-receptor for the GDNF family in neuronal cells. Expression of GDNF and GFRA1 has previously been found in human glioblastoma tissue samples, and in experimental studies, GDNF and GFRA1 have been shown to increase proliferation, migration, angiogenesis and increase chemoresistance in glioblastoma cells.

The overall aim of this PhD thesis was to investigate the expression of GDNF, the GDNF family of receptors and RET in human glioblastoma and the prognostic potential of GFRA1 and GFRA2 in glioblastoma. We also investigated the role of GDNF and GFRA1 in resistance to treatment with radiotherapy and chemotherapy in glioblastoma cells in order to investigate whether GDNF-GFRA1 signaling represents a therapeutic target for optimizing chemotherapy and radiotherapy for treatment of glioblastoma patients.

In Study I, the expression of GDNF, the GDNF family receptors GFRA1-4 and the RET receptor were investigated in human glioblastoma tissue samples. Varying levels of GDNF protein expression was present in frozen tumor tissue from 22 of the 27 patients investigated, while varying levels of GDNF mRNA was present in all of the formalin-fixed paraffin embedded (FFPE) tumor tissue from 10 patients investigated. Double stainings revealed high GDNF mRNA expression in GFAP-positive tumor cells and limited mRNA expression in IBA1-positive microglia and macrophages and CD34-positive endothelial cells. Receptor immunostainings showed strong expression of GFRA1, limited expression of GFRA2 and very low expression of GFRA3, GFRA4 and RET. Immunofluorescence double stainings showed strong GFRA1 expression in GFAP positive tumor cells and in less than 20 % of stem-like tumor cells positive for either OLIG2 or SOX2. GFRA1 expression was very low in IBA1-positive microglia and macrophages and absent in CD34-positive endothelial cells. The results suggest that GDNF mainly signal in an autocrine and paracrine manner to GFRA1-expressing tumor cells, including tumor stem cells. Furthermore, as RET expression was mostly absent, our results suggest that GDNF-GFRA1 signaling is RET-independent in glioblastoma.

In study II, the prognostic value of of GFRA1 and GFRA2 and co-expression with OLIG2 and IBA1, respectively, was further investigated. A fluorescent multiplex staining protocol was developed and stainings were performed on a glioblastoma patient cohort containing tumor tissue from 181 glioblastoma patients. The stained slides were scanned and spectrally unmixed digitally, and marker classifications and quantifications were performed. The results showed a wide range of GFRA1 expression. GFRA2 expression was only present in tumor tissue from a single patient. A small fraction of OLIG2 positive tumor stem cells also expressed GFRA1. Co-expression of GFRA1 and IBA1 was very low. There was no association between overall survival and expression of GFRA1 or co-expression of GFRA1 and OLIG2. Likewise, OLIG2 and IBA1 did not provide individual prognostic value. Even though GFRA1 was not associated with survival time, it was often expressed, and may still represent a therapeutic target for improved outcome for glioblastoma patients.

In study III, we investigated the role of GDNF and GFRA1 in resistance to treatment with either chemotherapy or irradiation in patient-derived glioblastoma cells. We saw an upregulation in mRNA expression of GDNF and GFRA1 after treatment with either temozolomide and lomustine, respectively, or irradiation. Using the CRISPR CAS9 technology, stable transfection models were created and validated for two of the cell lines with knockout of either GDNF or GFRA1. In the GDNF knock-out cell lines, the cells became sensitized to treatment with either temozolomide or lomustine, and exogenous addition of GDNF reversed the effect and desensitized the cells. GDNF knockout did not sensitize the cells to irradiation treatment. When GFRA1 was knocked out, the cells became sensitized to treatment with either temozolomide, lomustine or irradiation. The results suggest that GDNF and GFRA1 are mediators of resistance to chemotherapy, and that inhibition of GDNF-GFRA1 signaling may sensitize the tumor cells to chemotherapy treatment and irradiation treatment, and thus improve the efficacy of treatment in glioblastoma patients.

In conclusion, the studies presented in this thesis show that GDNF and GFRA1 are widely expressed in human glioblastomas, mainly in tumor cells. GDNF is also expressed in a small number of micro-glia and macrophages and endothelial cells, and GFRA1 is expressed in a subset of stem-like tumor cells. Due to very limited RET expression, we suggest that GDNF-GFRA1 signaling is RET-independent. GFRA1 expression did not carry prognostic value in glioblastoma patients. The results from the experimental part of the study suggest that GDNF-GFRA1 play a role in resistance to anti-neo-plastic treatment in glioblastomas. Further elucidating the mechanisms of GDNF-GFRA1 mediated treatment resistance in glioblastoma tumors can pave the way for targeted treatment that may increase the efficacy of the current standard treatment with chemotherapy and radiotherapy.