Exosomes secreted by ST3GAL5high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment.

Peritoneal dissemination of cancer affects patient survival. The behavior of peritoneal mesothelial cells (PMCs) and immune cells influences the establishment of a microenvironment that promotes cancer cell metastasis in the peritoneum. Here, we investigated the roles of lactosylceramide alpha-2,3-sialyltransferase (ST3G5; also known as ST3GAL5 and GM3 synthase) in the exosome-mediated premetastatic niche in peritoneal milky spots (MSs). Exosomes secreted from ST3G5high cancer cells (ST3G5high -cExos) were found to contain high levels of hypoxia-inducible factor 1-alpha (HIF1α) and accumulated in MSs via uptake in macrophages (MΦs) owing to increased expression of sialic acid-binding Ig-like lectin 1 (CD169; also known as SIGLEC1). ST3G5high -cExos induced pro-inflammatory cytokines and glucose metabolic changes in MΦs, and the interaction of these MΦs with PMCs promoted mesothelial-mesenchymal transition (MMT) in PMCs, thereby generating αSMA+ myofibroblasts. ST3G5high -cExos also increased the expression of immune checkpoint molecules and T-cell exhaustion in MSs, which accelerated metastasis to the omentum. These events were prevented following ST3G5 depletion in cancer cells. Mechanistically, ST3G5high -cExos upregulated chemokines, including CC-chemokine ligand 5 (CCL5), in recipient MΦs and dendritic cells (DCs), which induced MMT and immunosuppression via activation of signal transducer and activator of transcription 3 (STAT3). Maraviroc, a C-C chemokine receptor type 5 (CCR5) antagonist, prevented ST3G5high -cExo-mediated MMT, T-cell suppression, and metastasis in MSs. Our results suggest ST3G5 as a suitable therapeutic target for preventing cExo-mediated peritoneal dissemination.

CCDC85A is regulated by miR-224-3p and augments cancer cell resistance to endoplasmic reticulum stress.

MicroRNAs (miRNAs) play pivotal roles in the tumor microenvironment. Here, we analyzed miRNAs in tumor stromal fibroblasts. Expression of miR-224-3p in cancer-associated fibroblasts (CAF) from scirrhous gastric cancer patients was lower than in normal fibroblasts (NF). Introduction of a miR-224-3p mimic attenuated migration and invasion of CAF. Coiled-coil domain containing 85A (CCDC85A), whose function in tumors is not understood, was the target gene of miR-224-3p. Immunohistological analysis revealed that CCDC85A is expressed to varying degrees by cancer cells and CAFs in gastric and pancreatic carcinomas. Downregulation of CCDC85A in cancer cells revealed that these cells are vulnerable to endoplasmic reticulum (ER) stress induced by thapsigargin or tunicamycin, which were ameliorated after addback of CCDC85A. Injection of NF-derived exosomes containing miR-224-3p into the xenograft tumor increased tumor shrinkage by cisplatin treatment. Mechanistically, CCDC85A associated with the molecular chaperone GRP78 and GRP94, thereby inhibiting association of these negative regulators of the unfolded protein response (UPR), leading to sustained activation of PERK and downstream eIF2〈 and ATF4 upon ER stress. These data suggest a novel miR-224-3p-mediated function for CCDC85A: protection from ER stress and cisplatin resistance.

Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development.

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.

Pigment Epithelium Derived Factor Is Involved in the Late Phase of Osteosarcoma Metastasis by Increasing Extravasation and Cell-Cell Adhesion.

Organ tropism of metastatic cells is not well understood. To determine the key factors involved in the selection of a specific organ upon metastasis, we established metastatic cell lines and analyzed their homing to specific tissues. Toward this, 143B osteosarcoma cells were injected intracardially until the kidney-metastasizing sub-cell line Bkid was established, which significantly differed from the parental 143B cells. The candidate genes responsible for kidney metastasis were validated, and SerpinF1/Pigment epithelium derived factor (PEDF) was identified as the primary target. Bkid cells with PEDF knockdown injected intracardially did not metastasize to the kidneys. In contrast, PEDF overexpressing 143B cells injected into femur metastasized to the lungs and kidneys. PEDF triggered mesenchymal-to-epithelial transition (MET) in vitro as well as in vivo. Based on these results, we hypothesized that the MET might be a potential barrier to extravasation. PEDF overexpression in various osteosarcoma cell lines increased their extravasation to the kidneys and lungs. Moreover, when cultured close to the renal endothelial cell line TKD2, Bkid cells disturbed the TKD2 layer and hindered wound healing via the PEDF-laminin receptor (lamR) axis. Furthermore, novel interactions were observed among PEDF, lamR, lysyl oxidase-like 1 (Loxl1), and SNAI3 (Snail-like transcription factor) during endothelial-to-mesenchymal transition (EndoMT). Collectively, our results show that PEDF induces cancer cell extravasation by increasing the permeability of kidney and lung vasculature acting via lamR and its downstream genes. We also speculate that PEDF promotes extravasation via inhibiting EndoMT, and this warrants investigation in future studies.

SKAP2 suppresses inflammation-mediated tumorigenesis by regulating SHP-1 and SHP-2.

Inflammatory bowel diseases, like ulcerative colitis and Crohn's disease are frequently accompanied by colorectal cancers. However, the mechanisms underlying colitis-associated cancers are not fully understood. Src Kinase Associated Phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages. Here, we examined the effects of SKAP2 on inflammatory responses in a mouse model of tumorigenesis with colitis induced by azoxymethane/dextran sulfate sodium. SKAP2 knockout increased the severity of colitis and tumorigenesis, as well as lipopolysaccharide (LPS) induced acute inflammation. SKAP2 attenuated inflammatory signaling in macrophages induced by uptake of cancer cell-derived exosomes. SKAP2-/- mice were characterized by the activation of NF-κB signaling and the upregulation and release of cytokines including TNFα, IL-1ß, IL-6, CXCL-9/-10/-13, and sICAM1; SKAP2 overexpression attenuated NF-κB activation. Mechanistically, SKAP2 formed a complex with the SHP-1 tyrosine phosphatase via association with the Sirpα transmembrane receptor. SKAP2 also physically associated with the TIR domain of MyD88, TIRAP, and TRAM, adaptors of toll-like receptor 4 (TLR4). SKAP2-mediated recruitment of the Sirpα/SHP-1 complex to TLR4 attenuated inflammatory responses, whereas direct interaction of SKAP2 with SHP-2 decreased SHP-2 activation. SHP-2 is required for efficient NF-κB activation and suppresses the TRAM/TRIF-INFß pathway; therefore, SKAP2-mediated SHP-2 inhibition affected two signaling axes from TLR4. The present findings indicate that SKAP2 prevents excess inflammation by inhibiting the TLR4-NF-κB pathway, and it activates the TLR4-IFNß pathway through SHP-1 and SHP-2, thereby suppressing inflammation-mediated tumorigenesis.

Cancer-associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T-cell suppression.

In some tumors, a small number of cancer cells are scattered in a large fibrotic stroma. Here, we demonstrate a novel mechanism for expansion of pro-tumor fibroblasts via cancer-associated fibroblast (CAF)-mediated education of normal fibroblasts (NFs). When NFs were incubated with conditioned medium from CAFs, the resulting CAF-educated fibroblasts (CEFs) generated reactive oxygen species, which induced NF-κB-mediated expression of inflammatory cytokines and the extracellular matrix protein asporin (ASPN), while expression of a common CAF marker gene, α-SMA, was not increased. ASPN further increased CEF expression of downstream molecules, including indoleamine 2,3-dioxygenase 1 (IDO-1), kynureninase (KYNU), and pregnancy-associated plasma protein-A (PAPP-A). These CEFs induce cytocidal effects against CD8+ T cells and IGF-I activation in cancer cells. CEFs were generated without cancer cells by the direct mixture of NFs and CAFs in mouse xenografts, and once CEFs were generated, they sequentially educated NFs, leading to continuous generation of CEFs. In diffuse-type gastric cancers, ASPNhigh /IDO-1high /KYNUhigh /α-SMA- CEFs were located at the distal invading front. These CEFs expanded in the fibrotic stroma and caused dissemination of cancer cells. ASPN may therefore be a key molecule in facilitating tumor spreading and T-cell suppression.

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress.

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH-E1α, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN-/- mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

Macrophage-mediated transfer of cancer-derived components to stromal cells contributes to establishment of a pro-tumor microenvironment.

Tumor-derived extracellular vesicles (TEVs) secreted into the blood create a pre-metastatic niche in distant organs; however, it is unclear how TEVs are delivered and how they affect stromal cells in the tumor microenvironment. Tumor-associated macrophages (TAMs) have pivotal roles in cancer progression by interacting with cancer cells and other stromal cells. Here, we report a novel function of TAMs: delivery and transmission of TEV contents. TEV-incorporating macrophages (TEV-MΦs) showed increased invasiveness and were disseminated widely. Upon contact with host stromal cells (peritoneal mesothelial cells (PMCs), fibroblasts, and endothelial cells), TEV-MΦs released membrane blebs containing TEVs, a process dependent upon localized activation of caspase-3 in MΦs. Scattered blebs were incorporated into stromal cells, leading to transfer of cancer-derived RNA and proteins such as TGF-ß, activated Src, Wnt3, and HIF1α. TEV-MΦ-secreted blebs containing cancer-derived components contributed to myofibroblastic changes in recipient stromal cells. TEVs delivered by MΦs penetrated deep into the parenchyma of the stomach in TEV-injected mice, and transmitted TEVs to PMCs lining the stomach surface; this process induced PMCs to undergo mesothelial-mesenchymal transition. PMCs infiltrated the gastric wall and created a niche, thereby promoting tumor invasion. Depletion of MΦs prevented these events. Moreover, TEV-MΦs created a pro-metastatic niche. Taken together, these results suggest a novel function for TAMs: transfer of cancer-derived components to surrounding stromal cells and induction of a pro-tumor microenvironment via an increase in the number of CAF-like cells.

Curcumin analog, GO-Y078, overcomes resistance to tumor angiogenesis inhibitors.

Tumor angiogenesis inhibition is one of the most potent strategies in cancer chemotherapy. From past clinical studies, inhibition of the vascular endothelial growth factor pathway successfully treats malignant tumors. However, vascular endothelial growth factor inhibitors alone cannot cure tumors. Moreover, resistance to small molecule inhibitors has also been reported. Herein, we show the antiangiogenic potential of a newly synthesized curcumin analog, GO-Y078, that possibly functions through inhibition of actin stress fiber formation, resulting in mobility inhibition; this mechanism is different from that of vascular endothelial growth factor inhibition. In addition, we examined the detailed mechanism of action of the antiangiogenesis potential of GO-Y078 using human umbilical venous epithelial cells resistant to angiogenesis inhibitors (HUVEC-R). GO-Y078 inhibited the growth and mobility of HUVEC-R at 0.75 µmol/L concentration. Expression analyses by microarray and RT-PCR showed that expressions of genes including that of fibronectin 1 were significantly suppressed. Among these genes, fibronectin 1 is abundantly expressed and, therefore, seems to be a good target for GO-Y078. In a knockdown experiment using Si-oligo of fibronectin 1 (FN1), FN1 expression was decreased to half of that in mock experiments as well as GO-Y078. Knockdown of FN1 resulted in the suppression of HUVEC-R growth at 24 hours after treatment. Fibronectin is a key molecule contributing to angiogenesis that could be inhibited by GO-Y078. Thus, resistance to vascular endothelial growth factor inhibition can be overcome using GO-Y078.

Author Correction: Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

PLEKHN1 promotes apoptosis by enhancing Bax-Bak hetro-oligomerization through interaction with Bid in human colon cancer.

The anti-apoptotic nature of cancer cells often impedes the effects of anti-cancer therapeutic agents. Multiple death signals influence mitochondria during apoptosis, and though many studies have attempted to elucidate these complicated pathways, Bax oligomerization, an important step in the process, remains controversial. Here we demonstrate that pleckstrin-homology N1 (PLEKHN1), also known as cardiolipin phosphatidic acid binding protein, plays pro-apoptotic roles during reactive oxygen species (ROS)-induced apoptosis. Human PLEKHN1 was expressed in several cancer cell lines of differing origin. Its expression was regulated by hypoxia, and it existed in the mitochondrial fraction. Genome editing of hPLEKHN1 in human colon cancer HT-29 cells revealed enhanced survival of knockout cells compared with that of parental cells in vitro and in vivo. Thapsigargin or hydrogen peroxide treatment activated multiple death signals including JNK, Bcl-2 family members, and caspases. PLEKHN1 was bound to Bid, a pro-apoptotic protein, and not to Bax, and PLEKHN1 could remove Bid from transient Bid-Bax complexes. Fluorescent time-lapse imaging revealed that PLEKHN1 aggregated with Bid during thapsigargin- or hydrogen peroxide-induced apoptosis prior to Bax aggregation. Inhibition of PLEKHN1 led to attenuation of Bax-Bak hetero-oligomerization and Bid translocation. The immunohistochemistry of cancer patient specimens showed that PLEKHN1 expression was absent from cancer region at the transition area of normal/cancer tissues. Collectively, the silencing of PLEKHN1 may be the key that cancer cells acquire the drug resistance.

Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment.

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

The low expression of miR-451 predicts a worse prognosis in non-small cell lung cancer cases.

PURPOSE: miR-451 is a tumor suppressive microRNA with several target genes, including Macrophage migration inhibitory factor (MIF). As little is known about the expression and clinicopathological significance of mir-451 in NSCLC, we performed a clinicopathological study of 370 NSCLC cases to clarify them. Cell biological experiments were also performed on NSCLC cell lines to confirm the tumor-suppressive role of miR-451 and whether or not MIF is targeted by miR-451. METHODS: We analyzed 370 NSCLC cases for the miR-451 expression by quantitative real-time polymerase chain reaction and the MIF expression by immunohistochemistry. Eighty-four background lung tissue samples were also evaluated for the miR-451 expression. The clinicopathological and genetic factors surveyed were the disease-free survival, smoking status, histological type, disease stage, EGFR gene mutations and ALK rearrangements. In 286 adenocarcinoma cases, the invasive status (adenocarcinoma in situ, minimally invasive adenocarcinoma and invasive adenocarcinoma) was also evaluated. Five NSCLC cell lines (H23, H441, H522, H1703, and H1975) were cultured and evaluated for their miR-451 and MIF expression. The cell lines with lower miR-451 and higher MIF expressions were then selected and transfected with miR-451-mimic to observe its effects on MIF expression, Akt and Erk status, cell proliferation, and cell migration. RESULTS: The miR-451 expression was down-regulated in cancer tissues compared with background lung tissues (P<0.0001). Factors such as advanced disease stage, positive pleural invasion and nodal status and being a smoker were significantly correlated with a lower expression of miR-451 (P<0.05 each), while EGFR gene mutations and ALK rearrangements were not. In adenocarcinoma, invasive and minimally invasive adenocarcinoma showed lower expression of miR-451 than adenocarcinoma in situ (P<0.0005, respectively). A survival analysis showed that a lower expression of miR-451 was an independent predictor of a poor prognosis for NSCLC (P<0.05). The MIF expression was inversely correlated with the miR-451 expression. Out of 5 NSCLC cell lines examined, H441 and H1975 showed higher MIF and lower miR-451 expressions. After the transfection of miR-451-mimic, the MIF expression and phosphorylated Akt expression of these cell lines was suppressed, as were cell proliferation and cell migration. CONCLUSION: This clinicopathological study of 370 NSCLC cases and the cell biological studies of NSCLC cell lines clarified the tumor-suppressive role of miR-451 and its prognostic value. We also validated MIF as a target of miR-451 in NSCLC.

Spatiotemporal expression of UPK3B and its promoter activity during embryogenesis and spermatogenesis.

Uroplakin (Upk) 3 is one of the main structural components of the urothelium tissue. Although expression of UPK3B is seen in a wider variety of the tissues and organs than UPK3A, tissue-specific expression has not yet been analyzed. Here, we analyzed the Cre recombinase activity driven by the Upk3b promoter in transgenic mice and the endogenous localization of UPK3B. We generated Tg(Upk3b-Cre)/R26tdTomato mice by crossing ROSA26tm14(CAG-tdTomato) (R26tdTomato) mice with Tg(Upk3b-Cre) mice and investigated the spatiotemporal distribution of tdTomato in embryonic and adult mice. In embryos, we detected Cre recombinase activity in neural crest cells and the heart, liver, kidneys, and lungs. In adult mice, Cre recombinase activity was detected in male and female genital organs; however, the activity was absent in the bladder. Histological analyses revealed that both tdTomato and UPK3B were present in testicular and epididymal sperm; however, tdTomato was not present in the ductus epididymis, where the endogenous expression of UPK3B was detected. In female siblings, both tdTomato and UPK3B expressions were detected in the follicles of the ovary, whereas no tdTomato expression was found in the mucosal epithelium of the fallopian tubes, where the endogenous UPK3B was expressed. These data suggest that UPK3B may play a pivotal role in the maturation of gametes and gamete-delivery organs.

Mesothelial Cells Create a Novel Tissue Niche That Facilitates Gastric Cancer Invasion.

Peritoneal mesothelial cells (PMC) cover organ surfaces in the abdominal cavity. In this study, lineage tracing revealed that the PMCs guide cancer cell invasion in the gastric wall and in peritoneal metastatic lesions. Serosal PMCs covering the stomach surface entered the gastric wall to create a novel niche that favored gastric cancer cell invasion. PMC infiltration was induced by incorporation of cancer cell-derived, Wnt3a-containing extracellular vesicles. Infiltrated PMCs in turn promoted subserosal invasion of cancer cells. Mutual attraction between cancer cells and PMCs accelerated tumor invasion in the gastric wall, and PMC-led cancer cell invasion in disseminated tumors within the abdominal wall and diaphragm. Addition of the carboxyl terminus of Dickkopf-1 attenuated directional invasion of PMCs toward cancer cells both in vitro and in the gastric wall in vivo PMCs were sensitive to the aldehyde dehydrogenase (ALDH) inhibitor disulfiram (DSF), as ALDH activity is elevated in PMCs. Wnt3a upregulated ALDH, and addition of DSF inhibited the invasive properties of PMCs, whereas DSF pretreatment suppressed gastric infiltration of PMCs and subserosal invasion by cancer cells. Our results suggest that stabilization of PMCs may become an effective therapy for the prevention of local invasion and metastasis of gastric cancer. Cancer Res; 77(3); 684-95. ©2016 AACR.

Identification of anti-cancer chemical compounds using Xenopus embryos.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

SKAP2 Promotes Podosome Formation to Facilitate Tumor-Associated Macrophage Infiltration and Metastatic Progression.

Tumor-associated macrophages (TAM) play complex and pivotal roles during cancer progression. A subset of metastasis-associated macrophages accumulates within metastatic sites to promote the invasion and growth of tumor cells. Src kinase-associated phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages from various tumors, but its contribution to the tumor-promoting behavior of TAMs is unknown. Here, we report that SKAP2 regulates podosome formation in macrophages to promote tumor invasion and metastasis. SKAP2 physically interacted with Wiskott-Aldrich syndrome protein (WASP) and localized to podosomes, which were rarely observed in SKAP2-null macrophages. The invasion of peritoneal macrophages derived from SKAP2-null mice was significantly reduced compared with wild-type macrophages, but could be rescued by the restoration of functional SKAP2 containing an intact tyrosine phosphorylation site and the ability to interact with WASP. Furthermore, SKAP2-null mice inoculated with lung cancer cells exhibited markedly decreased lung metastases characterized by reduced macrophage infiltration compared with wild-type mice. Moreover, intravenously injected SKAP2-null macrophages failed to efficiently infiltrate established tumors and promote their growth. Taken together, these findings reveal a novel mechanism by which macrophages assemble the appropriate motile machinery to infiltrate tumors and promote disease progression, and implicate SKAP2 as an attractive candidate for therapeutically targeting TAMs.

A Curcumin Analog, GO-Y078, Effectively Inhibits Angiogenesis through Actin Disorganization.

BACKGROUND: The inhibition of angiogenesis is a theoretically ideal chemotherapy for cancer, but there remains room for improvement. Most inhibitors of angiogenesis approved to date target vascular endothelial growth factors (VEGFs); however, VEGFs are only one of the many classes of participant in tumor angiogenesis. Because tumor angiogenesis is orchestrated by many components, including growth factors, signal transducers, and effectors, its regulation exhibits redundancy. Curcumin can associate with many proteins, and it reportedly inhibits tumor angiogenesis. OBJECTIVE: We investigated the ability of a new curcumin analog, GO-Y078, to inhibit tumor angiogenesis. RESULTS: GO-Y078 inhibited human umbilical vascular endothelial cell sprouting. GO-Y078 also induced complete anoikis in vascular endothelial cells. Moreover, GO-Y078 suppressed the migration and invasion of vascular endothelial cells into extracellular matrix proteins. However, expression analysis revealed that GO-Y078 did not suppress molecules involved in VEGF signaling. Rather, GOY078 induced actin disorganization, dissociation of vinculin from actin, and destruction of focal adhesion, resulting in the inhibition of vascular endothelial cell mobility. GO-Y078 also suppressed in-vivo vasculogenesis in Xenopus laevis tadpoles. CONCLUSION: Actin organization is a common effecter related to vascular endothelial cell mobility in angiogenesis. We demonstrated that GO-Y078 inhibits angiogenesis through actin disorganization.

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity.

Collective cell migration (CCM) and epithelial-mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell-cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell-cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like-to-fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness.

Nm23-H1 regulates contact inhibition of locomotion, which is affected by ephrin-B1.

Contact inhibition of locomotion (CIL) is the process by which cells stop the continual migration in the same direction after collision with another cell. Highly invasive malignant cells exhibit diminished CIL when they contact stromal cells, which allows invasion of the tissue by tumors. We show that Nm23-H1 is essential for the suppression of Rac1 through inactivation of Tiam1 at the sites of cell-cell contact, which plays a pivotal role in CIL. U87MG cells show CIL when they contact normal glia. In spheroid confrontation assays U87MG cells showed only limited invasion of the glial population, but reduction of Nm23-H1 expression in U87MG cells abrogated CIL resulting in invasion. In U87MG cells, Nm23-H1 is translocated to the sites of contact with glia through association with α-catenin and N-cadherin. Mutants of Nm23-H1, which lacked the binding ability with Tiam1, or α-catenin did not restore CIL. Moreover, the expression of ephrin-B1 in tumor cells disrupted CIL and promoted invasion. As one mechanism, ephrin-B1 inhibits the association of Nm23-H1 with Tiam1, which contributes for activation of Rac1. These results indicate a novel function of Nm23-H1 to control CIL, and its negative regulation by ephrin-B1.