Congress Abstracts - Society for Developmental Biology

Program/Abstract # 431
Oncogenic K-Ras promotes basal extrusion of epithelial cells by degrading S1P through autophagy
Slattum, Gloria Mercedes; Gu, Yapeng; Rosenblatt, Jody (University of Utah, USA)
Epithelia provide a protective barrier for the organs they encase, yet the cells compromising the epithelia are constantly turning over
via cell death and cell division. To maintain a protective barrier during tissue development and homeostasis, epithelia extrude cells
destined to die by contracting a band of actin and myosin. Although extrusion can remove cells triggered to die by apoptotic stimuli,
during homeostasis, epithelia extrude live cells, which then die by anoikis. Because transformed cells may override anoikis and
survive after extrusion, the direction of extrusion has important consequences for the extruded cell's fate. As most cells extrude
apically, they are eliminated through the lumen, however, cells with upregulated survival signals that extrude basally could potentially
invade the underlying tissue and migrate to other sites in the body. We found oncogenic K-Ras cells extruded basally, rather than
apically, in a cell-autonomous manner and can survive and proliferate following extrusion. Expressing oncogenic K-Ras V12 downregulates
the bioactive lipid Sphingosine 1 Phosphate (S1P) and its receptor S1P 2 , both of which are required for apical extrusion.
Surprisingly, the S1P biosynthetic pathway is not affected, as the S1P precursor, sphingosine kinase, and the degradative enzymes S1P
lyase and S1PP phosphatase are not significantly altered. Instead, we found that S1P is degraded by autophagy, which is highly
pronounced in extruding Ras V12 cells. Disruption of autophagy chemically or genetically in K-Ras V12 cells rescues S1P localization
and apical extrusion. We propose that basal cell extrusion provides a novel mechanism for cells to exit the epithelium and initiate
invasion into the surrounding tissues.
Program/Abstract # 432
Akt-p53-miR-365-cyclin D1/cdc25A Axis Contributes to Gastric Tumorigenesis Induced by PTEN Deficiency
Teng, Yan; Yang, Xiao (Institute of Biotechnology, China)
Although PTEN/Akt signaling is frequently deregulated in human gastric cancers, the in vivo causal link between its dysregulation
and gastric tumorigenesis has not been established. Here we show that inactivation of PTEN in mouse gastric epithelium initiates
spontaneous carcinogenesis with complete penetrance by 2 months of age. Mechanistically, activation of Akt suppresses the
abundance of p53, leading to decreased transcription of miR-365, thus causing upregulation of cyclin D1 and cdc25A that promote
gastric cell proliferation. Importantly, genetic ablation of Akt1 restores miR-365 expression and effectively rescues gastric
tumorigenesis in PTEN-mutant mice. Moreover, orthotopic restoration of miR-365 represses PTEN-deficient-induced hyperplasia.
These data demonstrate that PTEN-Akt-p53-miR-365-cyclin D1/cdc25A axis serves as a new mechanism underlying gastric
tumorigenesis, providing novel potential therapeutic targets.
Program/Abstract # 433
The ciliary localization of Gli2 is important for its activation by Hh
Liu, Aimin; Liu, Jinling; Zeng, Huiqing (Penn State, USA)
Hedgehog (Hh) family of signaling proteins regulates cell growth and differentiation and requires the primary cilium for their function
in mammals. The Gli family of transcription factors, major downstream effectors of the Hh signaling pathway, are localized to the tip
of the primary cilium; however, how this localization is regulated and whether it is important for Gli activation remain poorly
understood. By deletion and domain swapping experiments, we identified a part of the Gli2 protein as the ciliary localization domain,
or CLD. A Gli2 variant lacking this domain, Gli2dCLD, is not localized to the cilium, even in the presence of activated Smoothened,
or with defective retrograde intraflagellar transport. Our in vitro over-expression analysis indicated that Gli2dCLD retains intrinsic
transcriptional activity and remains responsive to the inhibition of Suppressor of Fused. To investigate whether Gli2 ciliary
localization is required for its activation in vivo, we replaced the endogenous Gli2 with Gli2dCLD in mouse through gene-targeting.
The homozygous Gli2dCLD mutant embryos exhibit reduced expression of Hh target genes Ptch1 and Gli1, and defects in the spinal
cord patterning, suggesting that Gli2 activator activity is reduced. We found that the level of Gli2dCLD in the mutants is significantly
higher than that of Gli2 in wild type, suggesting that Gli2 activation, rather than its stability, is compromised in Gli2dCLD mutants.
Furthermore, the dampened Hh pathway activation in Gli2dCLD;Ptch1 double mutants suggests that Gli2 has to be in the cilium to
respond to upstream Hh pathway activation. In summary, our data shows that the full activation of Gli2 in vivo requires its ciliary
localization.
Program/Abstract # 434
KIF17 controls ciliary localization and function of GLI2
Carpenter, Brandon S.; Blasius, Teresa L.; Verhey, Kristen J. ; Allen, Benjamin L. (U Michigan- Ann Arbor, USA)
Primary cilia are cellular organelles that are essential for Hedgehog (HH) signal transduction during vertebrate embryogenesis. The
HH transcriptional effectors GLI2 and GLI3 traffic through primary cilia, which is required for proper processing of GLI proteins.
However, the mechanisms that control the ciliary targeting and trafficking of the GLI proteins are largely unknown. Kinesin-2 motor
proteins, namely KIF3A, KIF3B, and KIF17, mediate anterograde trafficking of proteins through primary cilia, making them
presumptive candidates for regulating anterograde transport of GLI2 and GLI3. However, since KIF3A and KIF3B function in both
anterograde cilia transport as well as in cilia formation, dissecting out a HH-specific function is difficult. Unlike KIF3A and KIF3B,
KIF17 function appears to be restricted to anterograde trafficking of cargo proteins and does not affect primary cilia formation. Here
we show that expression of dominant negative versions of KIF17 perturbs GLI2 and GLI3 ciliary localization. In addition,
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