TY - BOOK

T1 - Cellular Mechanisms Regulating Ciliary Disassembly and EMT: Roles of Ion Transport and Implications for Cancer

AU - Malinda, Raj Rajeshwar

PY - 2016

Y1 - 2016

N2 - Primary cilia are microtubule-based, antenna-like sensory organelles that emanate from the surface of most quiescent mammalian cells. Ciliary assembly and disassembly are tightly coupled with cell cycle progression. In cultured cells, cilia are formed in serum-deprived conditions (G0 or G1 phase) and disassemble upon cell cycle re-entrance (G1-S transition), stimulated by serum or growth-factors. Several previous studies have reported the possible involvement of platelet-derived growth factors (PDGFs) in ciliary resorption, but the specific receptor isoforms and mechanisms involved were unclear. In this thesis (Paper I), I show that primary cilia are dissembled upon PDGF-DD ligand mediated activation of PDGFRβ in cultured human rental pigment epithelial 1 (RPE1) cells. Furthermore, I demonstrate that PLCγ and intracellular Ca2+ release are key mediators of PDGFRβ induced deciliation. In contrast, PDGFAA mediated PDGFRα activation did not trigger ciliary disassembly, but expression of constitutively active PDGFRα mutant receptor D842V (work done by my colleague Brian Skriver Nielsen), which also potently activates PLCγ, induced significant deciliation in cultured cells. The ciliary disassembly phenotype of D842V expressing cells was restored upon inhibition of PDGFRα D842V kinase activity and AURKA kinase (AURKA). In summary, this study indicates that PDGFRβ and mutant PDGFRα D842V promote ciliary disassembly through PLCγ-mediated Ca2+ release, which in turn activates an AURKA-dependent disassembly pathway.Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancer types globally, is characterized by high levels of Transforming growth factor (TGF)β signaling and epithelial-tomesenchymal (EMT). In paper II, I show that TGFβ signaling elicits EMT in SMAD4-positive Panc-1 PDAC cells, as judged by the downregulation of E-cadherin and upregulation of α- smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). A modest induction of EMT was also observed in the SMAD-deficient BxPc-3 cells. TGFβ treatment increased Panc- 1 cell invasiveness about 5-fold, and significantly upregulated the expression of the acidextruding proteins NHE1 and NBCn1, and decreased that of the tumor suppressor merlin. Knockdown of merlin did not alter EMT induction per se, yet increased invasiveness and NHE1 expression. Knockdown of NHE1 and NBCn1 also did not alter EMT induction. Surprisingly, knockdown of NHE1 increased TGFβ-induced Panc-1 cell invasiveness, yet the invasiveness induced by merlin knockdown was abolished by knockdown of either NHE1 or NBCn1. Furthermore, TGFβ treatment increased proliferation in Panc-1 cells in a partially NHE1 dependent manner.In conclusion, the findings presented in paper I of this thesis provide new insight into the molecular mechanisms of ciliary disassembly by demonstrating a critical role for PDGFRmediated PLCγ- and Ca2+ signaling in this process. Findings presented in paper II show that acid extruding ion transporters are upregulated, while merlin is downregulated, during EMT in PDAC cells and regulate to the invasiveness and proliferation associated with the EMT phenotype.

AB - Primary cilia are microtubule-based, antenna-like sensory organelles that emanate from the surface of most quiescent mammalian cells. Ciliary assembly and disassembly are tightly coupled with cell cycle progression. In cultured cells, cilia are formed in serum-deprived conditions (G0 or G1 phase) and disassemble upon cell cycle re-entrance (G1-S transition), stimulated by serum or growth-factors. Several previous studies have reported the possible involvement of platelet-derived growth factors (PDGFs) in ciliary resorption, but the specific receptor isoforms and mechanisms involved were unclear. In this thesis (Paper I), I show that primary cilia are dissembled upon PDGF-DD ligand mediated activation of PDGFRβ in cultured human rental pigment epithelial 1 (RPE1) cells. Furthermore, I demonstrate that PLCγ and intracellular Ca2+ release are key mediators of PDGFRβ induced deciliation. In contrast, PDGFAA mediated PDGFRα activation did not trigger ciliary disassembly, but expression of constitutively active PDGFRα mutant receptor D842V (work done by my colleague Brian Skriver Nielsen), which also potently activates PLCγ, induced significant deciliation in cultured cells. The ciliary disassembly phenotype of D842V expressing cells was restored upon inhibition of PDGFRα D842V kinase activity and AURKA kinase (AURKA). In summary, this study indicates that PDGFRβ and mutant PDGFRα D842V promote ciliary disassembly through PLCγ-mediated Ca2+ release, which in turn activates an AURKA-dependent disassembly pathway.Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancer types globally, is characterized by high levels of Transforming growth factor (TGF)β signaling and epithelial-tomesenchymal (EMT). In paper II, I show that TGFβ signaling elicits EMT in SMAD4-positive Panc-1 PDAC cells, as judged by the downregulation of E-cadherin and upregulation of α- smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). A modest induction of EMT was also observed in the SMAD-deficient BxPc-3 cells. TGFβ treatment increased Panc- 1 cell invasiveness about 5-fold, and significantly upregulated the expression of the acidextruding proteins NHE1 and NBCn1, and decreased that of the tumor suppressor merlin. Knockdown of merlin did not alter EMT induction per se, yet increased invasiveness and NHE1 expression. Knockdown of NHE1 and NBCn1 also did not alter EMT induction. Surprisingly, knockdown of NHE1 increased TGFβ-induced Panc-1 cell invasiveness, yet the invasiveness induced by merlin knockdown was abolished by knockdown of either NHE1 or NBCn1. Furthermore, TGFβ treatment increased proliferation in Panc-1 cells in a partially NHE1 dependent manner.In conclusion, the findings presented in paper I of this thesis provide new insight into the molecular mechanisms of ciliary disassembly by demonstrating a critical role for PDGFRmediated PLCγ- and Ca2+ signaling in this process. Findings presented in paper II show that acid extruding ion transporters are upregulated, while merlin is downregulated, during EMT in PDAC cells and regulate to the invasiveness and proliferation associated with the EMT phenotype.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122244748705763

M3 - Ph.D. thesis

BT - Cellular Mechanisms Regulating Ciliary Disassembly and EMT: Roles of Ion Transport and Implications for Cancer

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -