The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells.

YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis.

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila.

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch-dedicated transcription factor. The Notch-dependent neoplasms require, however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1 and basic leucine zipper factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally, our work highlights some Notch/scrib specificities, in particular the role of the PAR domain-containing basic leucine zipper transcription factor and Notch direct target Pdp1 for neoplastic growth.

MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis.

Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.

Adherens Junction and E-Cadherin complex regulation by epithelial polarity.

E-Cadherin-based Adherens Junctions (AJs) are a defining feature of all epithelial sheets. Through the homophilic association of E-Cadherin molecules expressed on neighboring cells, they ensure intercellular adhesion amongst epithelial cells, and regulate many key aspects of epithelial biology. While their adhesive role requires these structures to remain stable, AJs are also extremely plastic. This plasticity allows for the adaptation of the cell to its changing environment: changes in neighbors after cell division, cell death, or cell movement, and changes in cell shape during differentiation. In this review we focus on the recent advances highlighting the critical role of the apico-basal polarity machinery, and in particular of the Par3/Bazooka scaffold, in the regulation and remodeling of AJs. We propose that by regulating key phosphorylation events on the core E-Cadherin complex components, Par3 and epithelial polarity promote meta-stable protein complexes governing the correct formation, localization, and functioning of AJ.

Dissecting the mechanisms of Notch induced hyperplasia.

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

The Drosophila GIPC homologue can modulate myosin based processes and planar cell polarity but is not essential for development.

Epithelia often show, in addition to the ubiquitous apico-basal (A/B) axis, a polarization within the plane of the epithelium, perpendicular to the A/B axis. Such planar cell polarity (PCP) is for example evident in the regular arrangement of the stereocilia in the cochlea of the mammalian inner ear or in (almost) all Drosophila adult external structures. GIPCs (GAIP interacting protein, C terminus) were first identified in mammals and bind to the Galphai GTPase activating protein RGS-GAIP. They have been proposed to act in a G-protein coupled complex controlling vesicular trafficking. Although GIPCs have been found to bind to numerous proteins including Frizzled receptors, which participate in PCP establishment, there is little in vivo evidence for the functional role(s) of GIPCs. We show here that overexpressed Drosophila dGIPC alters PCP generation in the wing. We were however unable to find any binding between dGIPC and the Drosophila receptors Fz1 and Fz2. The effect of overexpressed dGIPC is likely due to an effect on the actin cytoskeleton via myosins, since it is almost entirely suppressed by removing a genomic copy of the Myosin VI/jaguar gene. Surprisingly, although dGIPC can interfere with PCP generation and myosin based processes, the complete loss-of-function of dGIPC gives viable adults with no PCP or other detectable defects arguing for a non-essential role of dGIPC in viability and normal Drosophila development.

CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila.

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.

The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton.

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. Here, we have analyzed a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, we show that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. Our results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system.

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye.

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes.

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/./MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.

Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate beta-catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/beta-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/beta-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/beta-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/beta-catenin signal.

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate.

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).