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Department of Molecular Biology and Biochemistry
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Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University
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We aim to figure out that roles of signal transduction pathway in cellular growth, differentiation, motility, and polarity to understand their disruption-mediated diseases and develop novel diagnostic and therapeutic tools. Although we particularly focus on Wnt signaling pathway, we are also analyzing other pathways. We recognize the signal transduction itself as a biological system that links genotypes with phenotypes, and we will create new paradigm of life science research and the understanding of diseases. The following studies are under the way.

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Roles of Wnt signaling in fetal lung epithelial morphogenesisNew

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages.

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Post-translational modifications of Wnt3a, Wnt5a, and Wnt11

Wnts are glycan- and lipid-modified morphogens that are important for cellular responses, but glycan profiles of Wnts are not known. Mass-spectrometric analyses revealed that Wnt3a is modified with two high-mannose-type glycans (Asn87 and Asn298), that Wnt5a is modified with two high-mannose-type glycans (Asn114 and Asn326) and hybrid-type glycan (Asn312), and that Wnt11 is modified with complex-type glycan (Asn40) and two high-mannose-type glycans (Asn90 and Asn300). Wnt5a and Wnt11 are modified with palmitoleic acid at Ser244 and Ser215, respectively, which is necessary for the interaction with Wntless and Frizzled receptors, as well as Wnt3a.

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Selective regulation of Wnt signaling by receptor-mediated endocytosis

There are at least two Wnt signaling pathways dependent on or independent of β-catenin. A group of Wnts, including Wnt1 and Wnt3a, activate the β-catenin pathway. In this pathway, the binding of Wnt3a to Frizzled (Fz) and low density lipoprotein receptor-related protein 6 (LRP6) triggers the phosphorylation of LRP6 and induces the recruitment of the Axin complex to phosphorylated LRP6. The phosphorylation of LRP6 occurs in the lipid raft microdomain and that Wnt3a induces the internalization of Fz and LRP6 through caveolin-mediated endocytosis. Caveolin may act on the Axin complex to dissociate β-catenin from Axin, resulting in the accumulation of β-catenin. Another group of Wnts, including Wnt5a and Wnt11, activate the β-catenin-independent pathway, which primarily modulates cell movement, as initially observed during embryogenesis. In this pathway, Wnt5a and Wnt11 binds to receptor tyrosine kinase-like orphan receptor 2 (Ror2) and receptor-like tyrosine kinase (Ryk), respectively, in addition to Fz. They induce the internalization of receptors in a clathrin-dependent manner and activate the small G protein Rac. We are currently analyzing the molecular mechanisms how endocytic processes of receptors are involved in the specific activation of the β-catenin-dependent and -independent pathways.

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Physiological roles of the binding of Wnt and heparin sulfate proteoglycan or basement membrane proteins

Heparin sulfate proteoglycans (HSPGs) are localized mainly on the cell surface and in extracellular matrix (ECM). The major cell membrane HSPGs are the transmembrane syndecans (SDCs) and the glycosylphosphatidylinositol (GPI) anchored glypicans (GPCs). Although HSPGs affect the β-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. We found that GPC4 is present in both lipid raft and non-lipid raft microdomains and that GPC4 in lipid raft microdominas activates the Wnt3a-dependent β-catenin pathway and GPC4 in non-lipid raft microdomains activates the Wnt5a-dependent β-catenin-independent pathway. In contrast, SDCs appear in non-lipid raft microdomains. It was also reported that SDC4 binds to R-spondin3 and cooperates with Wnt5a and Fz7 to activate JNK in Xenopus embryos through clathrin-mediated endocytosis. These results suggest that GPC4 and SDC4 concentrate Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, which regulates distinct Wnt signaling. To further understand the mechanism of the efficient binding of Wnts to their receptors, we are investigating basement membrane proteins and HSPGs which interact with Wnts and regulate Wnt signaling pathway.

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Regulation of polarized sorting of Wnts and their receptors in MDCK cells

The mechanism underlying how Wnts and their receptors are sorted to their specific destinations is largely unknown. We examined the polarized sorting of Wnts and their receptors. The apical and basolateral secretion of Wnts are regulated by different mechanisms. The basolateral secretion of Wnt3a and Wnt5a required clathrin and adaptor protein 1 (AP-1) in polarized epithelial cells. On the other hand, glycosylation processing at Asn40 and galectin-3 are required for the apical secretion of Wnt11. Wntless (Wls) is essential for Wnt secretion, and is trafficked to the basolateral membranes by clathrin and AP-1 from the trans-Golgi network. Frizzled2 mainly localized to the basolateral membranes in MDCK cells. LRP6 and Ror2, which are receptors for Wnt3a and Wnt5a, respectively, were also trafficked to the basolateral membranes using clathrin and AP-1, but their sorting did not require Wls. The signaling pathways of Wnt3a and Wnt5a were strongly activated when both Wnts stimulate the basolateral side. These results indicate that the appropriate Wnt receptors are sorted to the same polarized fractions as the corresponding Wnts, thereby leading to efficient activation of their signals.

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Cancer cells secrete Wnt5b-associated exosomes

Wnt5b, a member of the same family of proteins as Wnt5a of which overexpression is associated with cancer aggressiveness, is suggested to be involved in cancer progression, however the details remain unclear. It was reported that some Wnts were secreted with extracellular microvesicles, such as exosomes, which were recovered in the 100,000 × g centrifugation precipitation fractions from conditioned medium (CM). Wnt5b released from PANC-1 pancreatic cancer cells or Caco-2 colon cancer cells transiently expressing Wnt5b, was recovered in precipitation fractions. However, some cancer cell lines, including HeLaS3 cervical cancer, A549 lung adenocarcinoma, and KKLS gastric cancer, expressed Wnt5a endogenously, and most Wnt5a secreted in CM was present in supernatant fractions.

To examine whether Wnt5b is indeed associated with exosomes, we generated a plasmid vector expressing hemagglutinin (HA)-tagged Wnt5b (HA-Wnt5b), by introducing HA tag sequence at position Arg36, located in a weakly conserved surface region opposite the Frizzled receptor binding site. HA-Wnt5b was recovered in precipitation fractions from CM of PANC-1 cells expressing HA-Wnt5b. Immunoelectron microscopy revealed that HA-Wnt5b is present on exosomes isolated from PANC-1 cells expressing HA-Wnt5b.

To examine endogenous Wnt5b activity in a PANC-1 cell, the WNT5B gene was successfully knocked out by a CRISPR/Cas9 system. Precipitation fractions from CM of PANC-1 cells showed Wnt5b activity, but those from WNT5B knockout (KO) PANC-1 cells dramatically decreased the activity. Exosomes isolated from wild-type PANC-1 cells, but not those from WNT5B KO PANC-1 cells, stimulated cell migration of and increased 5-ethynl-2’-deoxyuridine (EdU) incorporation into A549 cells. In Caco-2 cells expressing Wnt5b, knockdown of TGS101, which is a maker of exosomes, suppressed the secretion of Wnt5b-associated exosomes and inhibited Wnt5b-dependent cell proliferation.

Taken together, these results suggested that Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner.

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Molecular mechanisms by which growth factors including Wnt regulates cell adhesion, migration, and polarization

Cell polarization and the adhesion of a cell to a substrate are necessary for the cell to spread and migrate. When cells migrate, small focal adhesions, referred to as focal complexes at the cell periphery are formed. Upon the generation of tension, these focal complexes mature into large, organized adhesions, such as focal adhesions. These processes are regulated by small GTP-binding proteins Rac, Rho, and Cdc42. Although some growth factors such as EGF, VEGF, FGF, and Wnt regulate cell polarization, adhesion and migration, the molecular mechanism by which individual growth factors regulate these processes is poorly understood. We found that Wnt5a regulates cell migration and adhesion through the activation of focal adhesion kinase (FAK), a component of focal adhesion and Rac. Furthermore, we showed that dishevelled (Dvl), an essential component of Wnt signaling binds to adenomatous polyposis coli (APC), as microtubule (MT)-plus-end-binding protein at the leading edge of migrating cells, and both proteins associated with focal adhesion components. The binding of APC to Dvl is involved in stabilization of MTs at the cell periphery and Wnt5a-dependent focal adhesion turnover. We are currently analyzing the molecular mechanisms how other growth factors coordinately regulate cell adhesion, migration, and polarization.

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Roles of Wnt signaling in mitotic phases

Wnt signaling is known to promote G1/S progression through the stimulation of gene expression, but whether this signaling regulates mitotic progression is not clear.

(A) We found that Dvls phosphorylated by Polo-like kinase 1 (Plk1) forms a complex with APC and Wnt receptors, Fz and LRP6 and that their interaction is required for spindle orientation (a). Phosphorylation of Dvl2 by Plk1 is also involved in the microtubule (MT) kinetochore interaction. On the other hands, Wnt receptors are not required in this process (b). Dvl2 activates spindle assembly checkpoint in Plk1-mediated Dvl2 phosphorylation-dependent and Wnt-receptors-independent manner. Furthermore, Dvl2 is involved in the activation of Mps1 and recruitment of Bub1 and BubR1 at kinetochore (c). These results suggest that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus-ends and the spindle assembly checkpoint (SAC) in Plk1-dependent and -independent manners.

(B) In addition to the involvement of Dvl2 in the early mitotic phase via the β-catenin pathway, Dvl2 is localized to midbody in late mitotic phase. Through our observations, we proposed a model that 1) at early telophase, Dvl2 and Fz2 are recruited to the center of midbody, stembody, and Wnt5a signaling is involved in the recruitment of Dvl2 to the dark zone, which depends on microtubules. 2) Accumulated Fz2 and Dvl2 at stembody enhance the stabilization of midbody microtubules, which also contributes to their efficient recruitment to the midbody. 3) At late telophase Fz2 relocates to both sides of the dark zone. Wnt5a signal strengthens the binding of Fz2 and CHMP4B and stabilizes the proper localization of CHMP4B to both sides of stembody, leading to complete abscission. These findings suggest that both β-catenin-dependent and -independent pathways are required for progression of mitosis in temporal and spatial restricted manner.

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Prickle1 promotes focal adhesion disassembly with the CLASP–LL5β complex

Prickle is known to play roles in planar cell polarity including convergent extension and cell migration; however, the detailed mechanism by which Prickle regulates cellular functions is not well understood. Here, we show that Prickle1 regulates front-rear polarization and migration of gastric cancer MKN1 cells. Prickle1 preferentially accumulated at the cell retraction site in close proximity to paxillin at focal adhesions (FAs). Prickle1 dynamics were associated with those of paxillin during FA disassembly. Furthermore, Prickle1 was required for FA disassembly. CLASP and LL5β have been reported to form a complex surrounding focal adhesions and controls microtubule-dependent FA disassembly. Prickle1 bound to CLASP and LL5β, and was required for the LL5β-dependent accumulation of CLASP at the cell edge. Knockdown of CLASP and LL5β suppressed Prickle1-dependent cell polarization and migration. Prickle1 localized to the membrane through its C-terminal farnesyl moiety, and the membrane localization was necessary for Prickle1’s ability to regulate migration, bind to CLASP and LL5β, and promote microtubule targeting of FAs. Taken together, these results suggest that Prickle1 promotes FA disassembly during the retraction processes of cell polarization and migration.

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Regulation of epithelial morphogenesis by soluble growth factors and adhesion

It is important to understand how individual cells organize into multicellular tissues. Extracellular matrix and growth factors are essential for establishing and maintaining epithelial tube formation by controlling elongation, polarization, and branching. We are interested in how multicellular cell motility, polarity, proliferation, and differentiation are coordinately controlled at molecular and cellular level to form epithelial tubules. To address these questions, we are currently examining the mechanism of polarized cyst formation and analyzing the roles of soluble factors such as Wnt and EGF in branching morphogenesis by the use of three-dimensional culture of epithelial cells in vitro. In addition, using organs from embryo such as salivary and mammary glands, kidney, and lung, we will clarify 1) the mechanisms for collective cell elongation, polarization, and branching induced by specific growth factors and adhesion in each organ and 2) the common or different mechanisms in epithelial tube formation from various tissues.

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Regulation of tubular formation of IEC6 cells by P2Y2 receptor expression

We developed a new in vitro tubular formation assay using rat intestinal epithelial cells (IEC6 cells) in 3D Matrigel. IEC6 cells formed cysts in 3D Matrigel, and simultaneous stimulation with Wnt3a and EGF (Wnt3a/EGF) induced tubular formation. Using DNA microarray analyses, we have found that Wnt3a/EGF induces the expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) and Arl4c promotes tubular formation through the regulation of Rac and Rho activities. In this study, DNA microarray analyses also revealed that P2ry2 (P2Y2receptor (P2Y2R) gene) mRNA is expressed by Wnt3a/EGF during tube formation.

P2Y2R is a nucleotide-activated G-protein coupled receptor and binds to both purine and pyrimidine, ATP and UTP. Although P2Y2R regulates various cellular functions, for example, cotransmission and neuromodulation in nerve cells, the involvement of P2Y2R expression in epithelial tubulogenesis has not been previously studied. Then, we tried to clarify P2Y2R-mediated regulation of tubulogenesis induced by Wnt3a/EGF.

We examined the involvement of P2Y2R in epithelial tubulogenesis induced by Wnt3a/EGF. Knockdown of P2Y2R using siRNA suppressed Wnt3a/EGF-induced tubular formation, and P2Y2R-HA expression rescued the phenotypes. These results indicate that P2Y2R is required for tubular formation induced by Wnt3a/EGF.

We examined the involvement of P2Y2R ligands in epithelial tubulogenesis. A P2Y2R mutant that lacks nucleotide responsiveness rescued the phenotypes resulting from P2Y2R knockdown, suggesting that nucleotide-dependent responses are not required for P2Y2R functions in tubular formation.

To clarify ligand-independent functions of P2Y2R in tubulogenesis, we focused on the Arg-Gly-Asp (RGD) sequence of P2Y2R which has been shown to associate with integrins. A P2Y2R mutant that lacks the integrin-binding activity lost the ability to regulate tubular formation. These results indicate that binding of P2Y2R to integrins is required for tubular formation induced by Wnt3a/EGF.

Additionally, P2Y2R expression inhibited the formation of a complex between fibronectin which contains RGD sequences, and integrins. Appropriate inhibition of the interaction between fibronectin and integrins by the cyclic RGD peptide (RGDfV) and fibronectin siRNA enhanced, but a large amount of fibronectin suppressed Wnt3a/EGF-induced tubular formation.

The present study demonstrated P2Y2R expression induced by Wnt3a/EGF causes tubular formation of IEC6 cells by preventing the binding of integrins and fibronectin rather than mediating nucleotide responses.

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Regulation of developmental switching between "morphogenesis" and "differentiation"

Growth factor signaling is involved in the development of various tubular organs, but how signaling regulates organ early morphogenesis and late differentiation remains to be clarified. Here, we show how Wnt signaling controls epithelial tubular branching morphogenesis and functional differentiation using the salivary gland as a model. Experiments using genetically manipulated mice and organ cultures revealed that Wnt signaling at early stage (E12-E14) of submandibular salivary gland (SMG) development inhibits end bud morphogenesis and differentiation into proacini by suppressing KIT expression, and concomitantly increased the expression of distal progenitor markers such as Sox10 and KRT14. In addition, Wnt signaling at the SMG development early stage promoted end bud cell proliferation, leading to duct formation. In contrast, Wnt signaling loss at a late stage (E15-E18) of SMG development promoted end bud differentiation into proacini and suppressed duct formation. Our results suggested that Wnt signaling fine-tunes the timing of end bud morphogenesis and differentiation into proacini, in co-operation with KIT signaling.

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Initiation and promotion of cancer triggered by aberrant regulation of Wnt signaling

Mutations of genes involved in Wnt signaling have been reported to associate with many cancers. In particular, mutations of core components in the β-catenin pathway such as APC, β-catenin, and Axin have been well known to lead to aberrant activation of this pathway, resulting in various cancers, including colorectal and hepatocellular cancers. Recently several groups including my lab have reported that abnormalities of the β-catenin-independent pathway also associate with progression and aggressiveness in several cancers, including melanoma, lung, gastric, and prostate cancers. The β-catenin-independent pathway has several activities to regulate cytoskeletal rearrangements, cell polarity, and intracellular calcium-dependent signaling to stimulate cell migration and adhesion. However, the molecular mechanism how aberrant regulation of its signaling pathway contributes to cancer progression remains unclear. We are currently studying the molecular actions of Wnt5a, a representative ligand for the β-catenin-independent pathway, to lead to initiation and promotion of cancer.

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Development of diagnostic and therapeutic devises for Wnt5a signaling as a molecular target in cancer

We found that Wnt5a is involved in malignancy of some kind of human cancer including gastric and prostate cancers. Immunohistochmical studies showed that expression of Wnt5a is observed in about 30% of gastric and prostate cancer cases. Expression of Wnt5a was associated with aggressiveness and poor prognosis of gastric cancer. In addition, injection of polyclonal anti-Wnt5a antibody suppressed the frequency of tumor metastasis of gastric cancer cells. Although these findings suggested that Wnt5a could be a novel target for cancer therapy, there are no specific therapeutic devises for Wnt5a-mediated cancers. To identify specific therapeutic devises for Wnt5a signaling as a molecular target in cancer, we are trying to produce neutralizing monoclonal antibodies to Wnt5a. Furthermore, we are going to identify small molecules inhibiting the secretion of Wnt5a or the binding of Wnt5a to its receptors by using a high-throughput screening (HTS).

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Wnt5a promotes cancer cell proliferation by receptor-mediated endocytosis-independent mechanisms

Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. For instance, Wnt5a promoted invasion but not proliferation in some cancer cells such as KKLS gastric cancer cells. It was also reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. Wnt5a indeed stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells.

Although an anti-Wnt5a polyclonal neutralizing antibody was generated previously, the neutralizing activity of polyclonal antibodies varies among antibody lots. Therefore, the generation of a neutralizing monoclonal antibody against Wnt5a was necessary to control quality and quantity of the antibody. We generated an anti-Wnt5a monoclonal antibody by using a phage library. An anti-Wnt5a monoclonal antibody inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively.

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Relationship between Wnt5a signaling and inflammatory responses

Wnt5a signaling has been recently suggested to be involved in inflammatory diseases. It was shown that expression of Wnt5a and Fz5 can be induced through Toll-like receptors (TLR) stimulated by Mycobacterium tuberculosis (M. tuberculosis) or lipopolysaccharide (LPS) in macrophages, and Wnt5a signaling is involved in producing pro-inflammatory cytokines, such as interleukin (IL)-12 and IL-6. Wnt5a has been detected in granulomatous lesions in the lungs of patients with M. tuberculosis, bone marrow macrophages in septic patients, and macrophages accumulated within the intima of atherosclerotic patients. However, it is still unclear how Wnt5a regulates inflammatory responses physiologically and how its aberrant signaling leads to inflammatory diseases.

We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2. Removing Wnt5a or Ror2 in adult mice did not show clear phenotypes, seems to be healthy macroscopically. However, in these CKO mice, dextran sodium sulfate (DSS)-induced colitis were suppressed. It also attenuated the DSS-dependent increase in inflammatory cytokine gene expression and decreased interferon-γ (IFN-γ)-producing CD4+ Th1 cells in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients.

Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naïve CD4+ T cells to IFN-γ-producing CD4+ Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-γ, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-γ signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.

It has been reported that the β-catenin dependent pathway is activated in intestinal DCs and is required for the secretion of immunosuppressive cytokines. In contrast, our results suggested that the β-catenin independent pathway thorough Wnt5a-Ror2 signaling augmented intestinal inflammatory responses in DCs. Therefore, it is intriguing to speculate that in addition to the previously reported action of the β-catenin dependent pathway, which triggers immunosuppressive responses, Wnt signaling may also trigger inflammatory responses through the β-catenin independent pathway in intestinal DCs. Tumorigenesis has been shown to be associated with chronic inflammation, and Wnt5a signaling is also involved in tumor growth and metastasis. Therefore, Wnt5a signaling might be a therapeutic target for human diseases associated with inflammatory disorders, in addition to cancer in future.

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Wnt5a signal promotes apical and basal polarization of single epithelial cells

Epithelial cells are organized into multicellular sheets of tubules that form distinct apical and basolateral (AB) compartments, which is known as epithelial polarization. It is well known that ECM-mediated signaling is important for the establishment of epithelial polarity. However, physiological cues and molecular mechanisms for ECM-induced AB polarization of normal epithelial cells remain unclear. We show that rat intestinal epithelial cells (IEC6 cells) can polarize at a single cell level in Matrigel- and Wnt5a signaling-dependent manner. When IEC6 cells were seeded on Matrigel, single cells formed an F-actin cap on the upper cell surface, where apical markers were accumulated, and a basolateral marker was localized to the rest of the cell surface region. Rac1 activity in the non-cap region was higher than that in the cap region, whereas Rho activity increased towards the cap region. Wnt5a signaling activated and inhibited Rac1 and RhoA, respectively, independently through Tiam1 and p190RhoGAP-A, which formed a tertiary complex with Dishevelled. These results suggest that Wnt5a promotes the AB polarization of IEC6 cells through the regulation of Rac and Rho activities in a Matrigel adhesion-dependent manner. This study revealed that Wnt5a promotes the AB polarization of IEC6 cells through the spatial regulation of Rac and Rho in an a manner dependent on the adhesion to ECM.

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Arl4c expression promotes tumorigenesis in colorectal and lung cancers

Wnt/β-catenin and EGF/Ras signaling have been reported to associate with many human cancers, including colorectal and lung cancers. We recently found that ADP-ribosylation factor (ARF)-like 4c (Arl4c) expression induced by a combination of Wnt/β-catenin and EGF/Ras signaling in normal epithelial cells grown in three-dimensional culture promotes cellular migration and proliferation, resulting in formation of tubular structures. Although the process of epithelial morphogenesis resembles tumor formation in that both expand in the mensenchyme. The involvement of Arl4c in tumorigenesis has remained unclear. Immunohistochemical analysis showed that Arl4c was not observed in non-tumor regions, but was overexpressed in tumor lesions in 50 ~ 80% of colorectal and lung cancers. In several colorectal and lung cancer cell lines, Arl4c expression was upregulated through Wnt/β-catenin or Ras/MAPK signaling. Inhibition of Arl4c expression in HCT116 colorectal cancer cells and A549 lung adenocarcinoma cells reduced migration, invasion, and proliferation capabilities both in vitro and in vivo. Furthermore, direct injection of Arl4c siRNA into HCT116 cell-derived tumors (in vivo treatment with siRNA) inhibited tumor growth in immunodeficient mice. These findings suggested that Arl4c could be a novel marker for cancers and a molecular target for cancer therapy. We are currently developing new nucleic acid based medicine representing therapeutic target for cancers with Arl4c overexpression.

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Epigenetic upregulation of Arl4c expression, due to DNA hypomethylation in the 3'-untranslated region, in lung squamous cell carcinoma

We recently found that expression of ADP-ribosylation factor (ARF)-like 4c (Arl4c) was highly expressed in tumor lesions of colon and lung adenocarcinomas, where genetic alterations of Wnt/β-catenin and EGF/Ras pathways are frequently observed, and Arl4c expression promoted migration, invasion, and proliferation of cancer cells both in vitro and in vivo. However, it is unclear whether Arl4c is overexpressed in tumors where aberrant activation of growth factor signaling, such as Wnt/β-catenin or EGF/Ras pathways, does not occur. Immunohistochemical analysis showed that Arl4c was not observed in non-tumor regions, but was overexpressed in tumor lesions in 70 ~ 80% of lung and tongue squamous cell carcinoma (SCC). Knockout of Arl4c expression in NCI-H520 lung SCC cells and SAS tongue SCC cells reduced proliferation and migration capabilities in vitro. Although inhibition of Wnt/β-catenin or Ras/MAP kinase signaling did not decrease Arl4c expression in NCI-H520 cells, Arl4c DNA was clearly hypomethylated in the 3’-untranslated region (UTR). Ten-eleven translocation methylcytosine dioxygenase (TET) enzyme, which mediates DNA demethylation, was highly expressed in NCI-H520 cells. Knockout of TET family proteins (TET1-3) in NCI-H520 cells promoted DNA methylation in the 3’-UTR, leading to the decrease in Arl4c expression. The analyses with The Cancer Genome Atlas (TCGA) dataset (379 lung cancer cases) revealed that DNA methylation in the 3’-UTR of Arl4c gene was lower than in non-tumor regions in Arl4c-positive lung SCC. The results were consistent with our observations. These findings suggested that Arl4c contributes to oncogenesis as a result of its overexpression by multiple mechanisms and a molecular target for cancer therapy. We are currently developing new nucleic acid based medicine representing therapeutic target for cancers with Arl4c overexpression.

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Dkk1-CKAP4 signaling promotes tumorigenesis in pancreas and lung cancers

Dickkopf 1 (Dkk1) is a secretory protein and antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related proteins 6 (LRP6). Dkk1 is also suggested to regulate its own signaling to promote cancer cell proliferation, but the mechanism has not been understood. We screened Dkk1-binding proteins and identified cytoskeleton-associated protein 4 (CKAP4) as a novel Dkk1 receptor. Dkk1-CKAP4 signaling activated AKT through the formation of a complex between CKAP4 and phosphatidylinositol 3-kinase (PI3K), resulting in cancer cell proliferation.

In pancreatic ductal adenocarcinoma, Dkk1 and CKAP4 were strongly detected in 45/59 (76.3%) and 39/59 (66.1%) cases, respectively. In lung adenocarcinoma, 53/67 (79.1%) and 50/67 (74.6%) cases were positive for Dkk1 and CKAP4 expression, respectively. In lung squamous cell carcinoma cases, Dkk1 and CKAP4 were positive in 45/61 (73.8%) and 42/61 (68.9%) cases, respectively. Both proteins were minimally detected in non-tumor regions under our staining conditions. In pancreatic cancer cases, the 5-year survival was about 50% in Dkk1 and/or CKAP4 negative patients, whereas it was significantly reduced in both Dkk1 and CKAP4 positive patients. In lung cancer cases, both Dkk1 and CKAP4 expression was negatively correlated with relapse-free survival. These indicate that their simultaneous expression is negatively correlated with prognosis and relapse-free survival of pancreatic and lung cancers.

The role of Dkk1-CKAP4 signaling in tumorigenesis in vivo was investigated by subcutaneously implanting cancer cells into the flanks of immunodeficient mice. The volumes and weights of the xenograft tumors derived from S2-CP8 pancreatic and A549 lung cancer cells stably expressing CKAP4 shRNA were less than those of control tumors. The anti-CKAP4 antibody blocked the binding of Dkk1 to CKAP4, thereby suppressing AKT activity in cancer cells and xenograft tumor formation in immunodeficient mice. Thus, CKAP4 might represent a novel therapeutic target for cancers expressing Dkk1 and CKAP4. We are currently generating monoclonal antibodies that specifically block Dkk1-CKAP4 signaling and would like to develop a novel cancer therapy.

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Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University
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