ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (P = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging-based translational approaches. Mol Cancer Ther; 15(5); 1145-52. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Erlotinib Hydrochloride/pharmacokinetics , Erlotinib Hydrochloride/therapeutic use , Models, Biological , Pancreatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Monitoring , Humans , Magnetic Resonance Imaging , Mice , Mice, Transgenic , Neoplasm Grading , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/mortality , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Survival Analysis , Time Factors , Tissue DistributionABSTRACT
The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a regulatory factor of the cell cycle. Its transcriptional activation and protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca²âº-binding proteins involved in several biological processes, including cell cycle progression and signal transduction. In the present study, we show that down-regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It appears that a ubiquitin-independent proteasomal degradation process is involved in p21 degradation in S100A11 down-regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analysis of distinct signal transduction pathways revealed a disturbed phosphatidylinositol-3-kinase/Akt pathway after S100A11 knockdown. We determined that the glycogen synthase kinase-3, which is negatively regulated by phosphatidylinositol 3-kinase/Akt, was activated in cells possessing knocked-down S100A11 and appears to be involved in p21 protein destabilization. The application of a specific inhibitor of glycogen synthase kinase 3 resulted in an increase of the p21 protein level in S100A11 down-regulated HaCaT cells. Glycogen synthase kinase 3 is able to phosphorylate p21 at T57, which induces p21 proteasomal turnover. Mutation of the glycogen synthase kinase 3 site threonine 57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down-regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in the maintenance of p21 protein stability and appears to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Down-Regulation , Keratinocytes/metabolism , S100 Proteins/metabolism , Up-Regulation , Apoptosis/drug effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/agonists , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/genetics , Down-Regulation/drug effects , Gene Silencing , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/chemistry , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Humans , Keratinocytes/drug effects , Mutant Proteins/agonists , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Stability/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , S100 Proteins/antagonists & inhibitors , S100 Proteins/genetics , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation.