The role of atypical protein kinase C iota in glioblastoma multiforme

Glioblastoma multiforme is the most aggressive form of primary brain tumour occurring in adults. Despite aggressive multimodal therapy which includes surgery, radiation and chemotherapy it remains largely incurable and patients have a mean survival time of 12--14 months. Therefore the need for improved therapeutic strategies is imperative. Identifying molecular mechanisms involved in the development and progression of glioblastoma will not only improve our overall understanding of the disease but also identify potential targets that may be exploited therapeutically. The phosphoinositide 3-kinase (PI3K) pathway is often constitutively active in glioblastoma tumours due to overexpression and mutation of the epidermal growth factor receptor, and deletion/loss of function of the tumour suppressor PTEN. The atypical protein kinase Cs (PKCiota (iota) and PKCzeta (zeta)) have been shown to be activated downstream of PI3K and contribute to the malignant phenotype of several types of human cancers, including lung and colon cancer. However they have not been studied in glioblastoma. Our analysis identified that glioblastoma cell lines express PKCiota and have no detectable PKCzeta. PKCiota is activated in glioblastoma cells and its protein expression is increased compared to normal human astrocytes. To study the role of PKCiota in glioblastoma cells RNA interference was used to specifically reduce its expression. Depletion of PKCiota in glioblastoma cells decreased their proliferation, motility and invasiveness and partially enhanced their sensitivity to cisplatin-induced cell death. To identify potential mechanisms through which PKCiota promotes these malignant characteristics gene expression microarray analysis was performed on U87MG glioblastoma cells depleted of PKCiota. This analysis identified that PKCiota affects many classes of genes involved in a variety of cellular processes. Within the set of genes that were negatively regulated by PKCiota, glia maturation factor beta (GMFbeta) and ras homology family member B (RhoB) were investigated further. GMFbeta has been shown to enhance p38MAPK activation and signaling. The p38MAPK pathway has been previously identified as a key mediator of cisplatin cytotoxicity. Our results demonstrate that PKCiota suppresses cisplatin-induced p38MAPK activation causing an enhancement in resistance through the repression of GMFbeta. Overexpression of GMFbeta in glioblastoma cells causes an enhancement of p38MAPK activation and cell death in response to cisplatin treatment. PKCiota was also found to repress the expression of RhoB, which unlike its other Rho family members functions in the repression of tumourgenicity. Examination of RhoB repression by PKCiota showed that this significantly increases the motility and invasiveness of glioblastoma cells as overexpression of RhoB decreased these properties. Additionally, our results have identified a mutually antagonistic relationship between PKCiota activity and RhoB expression that may be a sensitive switch between a motile and non-motile phenotype. Lastly, live cell imaging of glioblastoma cells stably depleted of PKCiota revealed that it is involved in the dynamics of leading edge formation and plays a role in mitosis. This work has demonstrated that inhibiting PKCiota may be a useful therapeutic strategy for glioblastoma, either alone or in combination with other treatment modalities, such as chemotherapy or radiation, to improve the poor patient outcomes associated with this disease.