Background: Heterozygous germline AIP mutations can lead to young-onset invasive GH-secreting adenomas. There are no data available to explain the proliferative and invasive nature of AIP-mutation positive somatotrophinomas.
Methods: Cell viability (MTS assay), invasion (single cell fluorescence invasion assay) and migration assays (Boyden chambers) were used to further characterise the phenotype of AIP-silenced GH3 cells. Affymetrix gene expression-profiling was performed on AIP-silenced GH3 cells vs. scrambled control. Results were validated by RT-qPCR and Western blotting.
Results: Lentiviral AIP shRNA-silenced cells proliferated more quickly than controls by 48 hours (25% increase in absorption in MTS assay, P<0.0001). In the RNA profiling study the AIP-knockdown differential expressed gene list was enriched for transcripts encoding proteins regulating actin cytoskeleton-associated activities (e.g. CDC42, WASL, ARPC2/3, WASP), and thus cell migration and invasion. The small Rho GTPase CDC42 was up-regulated at the RNA (↑43%, P=0.0152) and protein level (↑57%, P=0.0156). GH3 cells were unable to migrate on a plain plastic surface. However, unlike NT shRNA transduced controls, AIP-silenced GH3 cells were able to migrate in response to collagen I and FCS-containing medium, in Boyden chambers coated with collagen IV. Collagen IV is known to be abundant in the pituitary capsule. AIP-silenced GH3 cells showed threefold increased adherence (P=0.029) and twofold increased invasion (P<0.0001) through type IV collagen matrix.
Conclusion: CDC42 is a small rho GTPase, which is upregulated in various cancers and is associated with lung cancer metastasis. We propose that AIP silencing leads to CDC42 upregulation and that this results in signalling through actin cytoskeleton pathways, promoting filopodia and invadopodia formation. Further, we suggest that the predilection for adherence, migration across and invasion through type IV collagen matrix is a factor in the increased invasiveness of AIP mutated tumours. CDC42 may represent a druggable target for treatment of atypical adenomas.