: AIP mutation-positive familial isolated pituitary adenoma is commonly diagnosed in young patients who have a poor prognosis due to large, treatment-resistant tumours. Microarray analysis carried out on AIP knockdown pituitary (GH3) cells and control cells, identified CDC42 as one of the genes that was up-regulated by loss of AIP protein. This small Rho GTPase activates MAPK signalling, suggesting it may contribute to the proliferative phenotype of AIP knockdown cells. Using a specific non-competitive inhibitor of CDC42, ML141, we studied elements of the MAPK-pathway ERK1/2 (proliferative), JNK and p38 (dependent on context may promote proliferation or apoptosis/senescence) in stably-transfected GH3 cells.
Method: GH3 cells were transduced with lentiviruses containing either an AIP or control shRNA, and stable clones expanded. Cell viability MTS assays and immunoblotting for CDC42 and for MAPK-pathway components (total&phospho-ERK1/2, JNK and p38) were quantified in the presence and absence of ML141.
Results: AIP silencing significantly elevated CDC42 RNA and protein levels in the stably-transfected cell line (RNA, P=0.0156, protein P=0.0152 vs non-targeted control). Cell viability was significantly increased and a non-significant rise in the phospho/total ERK1/2 ratio was observed. No change in p38 activation was observed. Based on dose-response curves, 20 μM of ML141 was used to determine the effect of CDC42 inhibition on cell viability and MAPK activation in the AIP-silenced and control cell lines. ML141 reduced cell viability in both (P<0.0001). It had no significant effect on ERK1/2 activation but did increase p38 activation: AIP knockdown (19× P=0.0286) and non-targeted cells (13× P=0.0286).
Conclusion: Up-regulation of the ERK-pathway could be responsible for the proliferative phenotype seen in AIP-silenced cells. ML141 is associated with an increase in p38 activation, suggesting that p38-dependent apoptosis/senescence might be responsible for the reduced cell viability. ML141 is a potential candidate for AIP mutation-positive pituitary adenoma treatment.