Essential role of MARCKS in cortical actin dynamics during gastrulation movements

Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements

PLOD2 is essential to functional activation of integrin β1 for invasion/metastasis in head and neck squamous cell carcinomas.

Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers

PODXL1 promotes metastasis of the pancreatic ductal adenocarcinoma by activating the C5aR/C5a axis from the tumor microenvironment

Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors

Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness

Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes

P120-Catenin Isoforms 1 and 3 Regulate Proliferation and Cell Cycle of Lung Cancer Cells via β-Catenin and Kaiso Respectively

<div><h3>Background</h3><p>The different mechanisms involved in p120-catenin (p120ctn) isoforms' 1/3 regulation of cell cycle progression are still not elucidated to date.</p> <h3>Methods and Findings</h3><p>We found that both cyclin D1 and cyclin E could be effectively restored by restitution of p120ctn-1A or p120ctn-3A in p120ctn depleted lung cancer cells. When the expression of cyclin D1 was blocked by co-transfection with siRNA-cyclin D1 in p120ctn depleted cells restoring p120ctn-1A or 3A, the expression of cyclin E was slightly decreased, not increased, implying that p120ctn isoforms 1 and 3 cannot up-regulate cyclin E directly but may do so through up-regulation of cyclin D1. Interestingly, overexpression of p120ctn-1A increased β-catenin and cyclin D1 expression, while co-transfection with siRNA targeting β-catenin abolishes the effect of p120ctn-1A on up-regulation of cyclin D1, suggesting a role of β-catenin in mediating p120ctn-1A's regulatory function on cyclin D1 expression. On the other hand, overexpression of p120ctn isoform 3A reduced nuclear Kaiso localization, thus decreasing the binding of Kaiso to KBS on the cyclin D1 promoter and thereby enhancing the expression of cyclin D1 gene by relieving the repressor effect of Kaiso. Because overexpressing NLS-p120ctn-3A (p120ctn-3A nuclear target localization plasmids) or inhibiting nuclear export of p120ctn-3 by Leptomycin B (LMB) caused translocation of Kaiso to the nucleus, it is plausible that the nuclear export of Kaiso is p120ctn-3-dependent.</p> <h3>Conclusions</h3><p>Our results suggest that p120ctn isoforms 1 and 3 up-regulate cyclin D1, and thereby cyclin E, resulting in the promotion of cell proliferation and cell cycle progression in lung cancer cells probably via different protein mediators, namely, β-catenin for isoform 1 and Kaiso, a negative transcriptional factor of cyclin D1, for isoform 3.</p> </div

Kaiso is a bimodal modulator for Wnt/β-catenin signaling

AbstractThe Wnt family of secreted ligands plays critical roles during embryonic development and tumorigenesis. Here we show that Kaiso, a dual specific DNA-binding protein, functions as a bimodal regulator of canonical Wnt signaling. Loss-of-function analysis of Kaiso abrogated Wnt-mediated reporter activity and axis duplication, whereas gain-of-function analysis of Kaiso dose-dependently resulted in synergistic and suppressive effects. Our analyses further suggest Kaiso can regulate TCF/LEF1-activity for these effects via modulating HDAC1 and β-catenin-complex formation. Our studies together provide insights into why Kaiso null mice display resistance to intestinal tumors when crossed onto an ApcMin/+ background.Stuctured summaryMINT-6823807: HDAC1 (uniprotkb:Q13547) physically interacts (MI:0218) with beta catenin (uniprotkb:P35222) by anti tag coimmunoprecipitation (MI:0007)MINT-6823820: axin (uniprotkb:O15169) physically interacts (MI:0218) with beta catenin (uniprotkb:P35222) by anti tag coimmunoprecipitation (MI:0007

PKCδ is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. We identified novel genes encoding δ-type PKC in the Xenopus EST databases. Loss of PKCδ function revealed that it was essential for convergent extension during gastrulation. We then examined the relationship between PKCδ and the Wnt pathway. PKCδ was translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKCδ function inhibited the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKCδ formed a complex with Dishevelled, and the activation of PKCδ by phorbol ester was sufficient for Dishevelled translocation and JNK activation. Thus, PKCδ plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled

Cytoskeletal regulation by Wnt signaling in Xenopus gastrulation movements

　Gastrulation is one of the most important developmental events for many multicellular organisms. In the amphibian embryos, mesodermal cells involute to the inside of the embryo and migrate along the blastocoel roof (BCR) to establish the three germ layer structure and body axes. These processes involve several morphogenetic cell movements including convergent extension. In convergent extension, cells are polarized, elongated mediolaterally, and then intercalated each other.　It is known that Wnt signaling pathways play an essential role in the regulation of convergent extension movements. Wnts are a family of secreted proteins that regulate many biological processes. Functional analyses in Xenopus suggest that the Wnt family can be divided into two functionally distinct groups. The first group of Wnts induces a secondary axis when ectopically expressed in Xenopus embryos. They activate the canonical Wnt/β-catenin pathway and induce transcription of target genes such as siamois and Xnr3. The second group of Wnts, which include Wnt5a and Wnt11, activates the noncanonical Wnt signaling pathway that controls morphogenetic cell movements. The noncanonical Wnt pathway branches into two cascades. One is the PCP (Planar Cell Polarity) pathway, and the other is the Wnt/Ca2+ pathway. Previous data showed that Wnt/PCP pathway has been implicated in the regulation of convergent extension. One of the PCP signaling components Dishevelled (Dsh) is essential for convergent extension. However, it is largely unknown how the PCP pathway regulates convergent extension.　Because active cell movements occur during convergent extension, the regulation of cytoskeletal dynamics may be important for the regulation of this process. It has been shown that Dsh activates Rho family small GTPases, suggesting that the PCP pathway may be involved in the cytoskeletal regulation. In order to investigate the regulatory mechanisms of actin cytoskeleton during gastrulation, He cloned a gene encoding an actin-binding protein MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) from a Xenopus embryonic cDNA library. MARCKS was first identified as a PKC (Protein Kinase C) substrate in mammalian cells. It attaches with the plasma membrane through N-terminal myristoylation. He showed that loss of MARCKS function by MO (Morpholino oligonucleotide) in Xenopus embryo induced a gastrulation defect phenotype without affecting mesoderm induction. To elucidate why MARCKS MO caused gastrulation defect, cell biological analyses were conducted. During convergent extension, MARCKS MO inhibited polarization and intercalation of mesodermal cells. He performed further observation at the cellular level. As a result, cell adhesion on fibronectin, protrusive activity of mesodermal cells and cortical actin formation in the cells were also inhibited by MARCKS MO. Furthermore, He found that activation of the PCP pathway promoted formation of filopodia- and lamellipodia-like structures in ectoderm explant cells, and MARCKS MO inhibited this activity. These results indicate that MARCKS regulates cortical actin dynamics, and it is requisite for the morphological processes regulated by the PCP signaling pathway. In addition, MARCKS MO also severely impaired neural tube closure without affecting the neural induction. It is consistent with the phenotype of mice deficient in MARCKS. MARCKS function may be conserved in vertebrates. Taken together, MARCKS is an essential molecule not only for gastrulation movements but also neural tube closure through controlling the cortical actin formation. These results are shown and discussed in Chapter 2.　It is known that Dsh is translocated to the plasma membrane in response to Wnt signaling in animal cap cells. In this thesis, He showed the bipolor localization of Dsh in mesodermal cells during convergent extension. These data indicate that the regulation of Dsh localization is important for the regulation of convergent extension. But its regulatory mechanism is unknown. Thus, He analyzed molecular mechanism to regulate Dsh localization and identified three proteins involved in the PCP pathway, PKCδ, Gα11 (G11) and Gαi1 (Gi1). First He identified PKCδ as an essential factor to regulate Dsh localization and showed that it physically interacted with Dsh. Loss of PKCδ function induced a gastrulation defective phenotype without affecting mesoderm induction. Confocal microscopic analyses revealed that both PKCδ and Dsh were translocated from the cytoplasm to the plasma membrane by Fz7 signaling. In addition, loss of PKCδ function reduced the signal-dependent Dsh translocation. These results indicate that PKCδ regulates Dsh localization under the control of Wnt signaling. Next, He focused on heterotrimeric G protein α subunits. Injections of antisense MOs against G11 or Gi1 caused a phenotype in the body axis elongation and/or gastrulation defect. In addition, these MOs inhibited elongation of DMZ explants. These results suggested these G proteins might be required for convergent extension. Thus, He investigated functions of these G proteins in the PCP pathway and found that Gi1 and G11 are necessary for the membrane localization of Dsh. G11 MO reduced both hyperphosphorylation of Dsh and the protrusive activity induced by the PCP pathway, whereas Gi1 MO did not. These data indicate that both Gi1 and G11 are required for the Dsh translocation, but these molecules may play distinct roles. These results are shown and discussed in Chapter 3.　This work has demonstrated that the PCP pathway regulates convergent extension movements through cytoskeletal regulation, and identified molecules essential for the intracellular signaling components in this pathway. These findings may contribute to understand the mechanisms of convergent extension movements and the other developmental processes in which the PCP pathway is involved