phosphothreonine and Pancreatic-Neoplasms

Epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) formation are key underlying causes that promote extensive metastasis, drug resistance, and tumor recurrence in highly lethal pancreatic cancer. The mechanisms leading to EMT and CSC phenotypes are not fully understood, which has hindered the development of effective targeted therapies capable of improving treatment outcomes in patients with pancreatic cancer.. We show a central role of Aurora kinase A (AURKA) in promoting EMT and CSC phenotypes via ALDH1A1, which was discovered as its direct substrate using an innovative chemical genetic screen. AURKA phosphorylates ALDH1A1 at three critical residues which exert a multifaceted regulation over its level, enzymatic activity, and quaternary structure. While all three phosphorylation sites contribute to its increased stability, T267 phosphorylation primarily regulates ALDH1A1 activity. AURKA-mediated phosphorylation rapidly dissociates tetrameric ALDH1A1 into a highly active monomeric species. ALDH1A1 also reciprocates and prevents AURKA degradation, thereby triggering a positive feedback activation loop which drives highly aggressive phenotypes in cancer. Phospho-resistant ALDH1A1 fully reverses EMT and CSC phenotypes, thus serving as dominant negative, which underscores the clinical significance of the AURKA-ALDH1A1 signaling axis in pancreatic cancer.. While increased levels and activity of ALDH1A1 are hallmarks of CSCs, the underlying molecular mechanism remains unclear. We show the first phosphorylation-dependent regulation of ALDH1A1, which increases its levels and activity via AURKA. Recent global phospho-proteomic screens have revealed increased phosphorylation of ALDH1A1 at the T267 site in human cancers and healthy liver tissues where ALDH1A1 is highly expressed and active, indicating that this regulation is likely crucial both in normal and diseased states. This is also the first study to demonstrate oligomer-dependent activity of ALDH1A1, signifying that targeting its oligomerization state may be an effective therapeutic approach for counteracting its protective functions in cancer. Finally, while AURKA inhibition provides a potent tool to reduce ALDH1A1 levels and activity, the reciprocal loop between them ensures that their concurrent inhibition will be highly synergistic when inhibiting tumorigenesis, chemoresistance, and metastasis in highly aggressive pancreatic cancer and beyond.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Aurora Kinase A; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Enzyme Stability; Epithelial-Mesenchymal Transition; Feedback, Physiological; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Phosphorylation; Phosphothreonine; Protein Multimerization; Protein Transport; Proteolysis; Retinal Dehydrogenase; Subcellular Fractions

Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.

Topics: Caspase 3; Catechin; Cell Line, Tumor; Cell Respiration; Checkpoint Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Electron Transport; Fibroblasts; Humans; Mitochondria; Pancreatic Neoplasms; Phosphorylation; Phosphothreonine; Radiation Tolerance; Radiation-Sensitizing Agents; Radiation, Ionizing

Although cytoskeletal proteins such as myosin II are implicated in the control of insulin secretion, their precise role is poorly understood. In other secretory cells, myosin II is predominantly regulated via the phosphorylation of the regulatory light chains (RLC). The current study was aimed at investigating RLC phosphorylation in beta-cells. In both the insulin-secreting cell line RINm5F and rat pancreatic islets, the RLC was basally phosphorylated on the myosin light chain kinase sites (Ser19/Thr18). Phosphorylation at these sites was not consistently increased by either metabolic stimuli (glyceraldehyde/glucose) or the depolarizing agent KCl. The RLC sites phosphorylated by protein kinase C (PKC) (Ser1/Ser2) were unphosphorylated in the basal state, not affected by nutrients or KCl, and only slightly increased by the PKC activator phorbol 12-myristate 13-acetate (PMA). Like the other insulin secretagogues, however, PMA did promote serine phosphorylation of the myosin heavy chain (MHC) in RINm5F cells. Phosphopeptide mapping suggested that the same peptide was phosphorylated under both PMA and glyceraldehyde stimulation, which further extends our previous study of the Ca2+-dependent phosphorylation of this protein (Wilson JR, Ludowyke RI, Biden TJ: Nutrient stimulation results in a rapid Ca2+-dependent threonine phosphorylation of myosin heavy chain in rat pancreatic islets and RINm5F cells. J Biol Chem 273:22729-22737, 1998). Overall, our results demonstrate that in RINm5F cells and rat pancreatic islets, MHC phosphorylation correlates better with insulin secretion than phosphorylation of the RLC. We therefore propose that in beta-cells, in contrast to other secretory cells, phosphorylation of the MHC is more important than that of the RLC for regulation of the myosin II protein complex during insulin secretion.

Topics: Animals; Cells, Cultured; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Myosin Heavy Chains; Myosin Light Chains; Myosin-Light-Chain Kinase; Pancreatic Neoplasms; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorylation; Phosphoserine; Phosphothreonine; Potassium Chloride; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured