Activation of cdk4/Cyclind D1 and the Associated Attenuation of Rb Function in Pancreatic Neuroendocrine Tumors (Pan-NETs)
Laura H Tang, David S Klimstra. Memorial Sloan Kettering Cancer Center, New York, NY
Background: Pan-NETs pose significant challenges in clinical management largely due to the lack of identifications of key oncogenic pathways in this neoplasm. Currently, there are no reliable biomarkers to predict the recurrence and progression and treatment option is limited for this disease. Although changes in genes of the retinoblastoma (Rb) pathway can lead to formation of Pan-NET in mice, genomic investigations in humans have not revealed Rb pathway mutations.
Design: We investigated abnormalities in the Rb pathway, including cdk4, cyclind D1 and Rb protein status, in Pan-NETs and identified significant genetic aberrations and associated functional consequence which culminated in attenuated Rb function via phosphorylation, which could be restored by a small molecule, PD 0332991 in Pan-NET cell lines.
Results: 1) Immunohistochemistry was carried out on tissue microarray constructed with 92 cases of well-differentiated Pan-NET. Cdk4 was overexpressed in 58% and Cydlin D1 in 68% of Pan-NETs; phospho-Rb was observed in 68% of tumors. There was a statistically significant correlation between phospho-Rb and cdk4/cyclin D1 protein expression (p=0.02).
2) Quantitative RT-PCR was performed using mRNA from fresh frozen samples of Pan-NETs. When compared with normal pancreas tissue, the cdk4 transcript was markedly increased ranging from 1.2 to 97 fold (mean 12.5±2.5). Increased cdk4 transcription correlated with cdk4 protein overexpression. Furthermore, cdk4 transcript was significantly higher (p<0.001) in non-functional Pan-NETs (mean 14.6±3.0) when compared with functional Pan-NETs (mean 3.8±0.5).
3) Real time PCR analysis revealed cdk4 to be amplified in a subset of tumors. FISH analysis revealed extra copies of cdk4 located in chromosome 12q.
4) Compound PD 0332991, a specific cdk4/6 inhibitor, effectively induced growth arrest at G1 phase cell cycle up to ten fold at low concentration (100 nM) and significantly decreased phosphorylated-Rb levels in two Pan-NET cell lines QGP1 and BON1 with IC50s of 60 nM and 130 nM, respectively.
Conclusions: We have characterized an attenuated Rb tumor suppressor pathway in pancreatic neuroendocrine tumors. The collective genomic, biologic and clinical substantiation suggest that combined cdk4 and cyclin D1 inhibition may prove to be an alternative beneficial therapeutic strategy for patients with this disease.
Monday, March 19, 2012 9:15 AM
Platform Session: Section G1, Monday Morning