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Diffuse-type gastric carcinoma is characterized by highly invasive growth, massive fibrosis, and peritoneal metastasis. Peritoneal metastasis is the leading cause of death in diffuse-type gastric carcinoma; however, the underlying molecular mechanisms remain largely unknown. Recent studies have established a concept of polyclonal metastasis, in which metastases arise from circulating tumor cell clusters with high metastatic potential. Polyclonal metastasis is a cause of tumor heterogeneity that contributes to tumor progression and therapeutic resistance. However, the clonality of peritoneally metastasized tumors and the evidence for polyclonal metastasis in diffuse-type gastric carcinoma have been poorly elucidated. In this study, we utilized multicolor fluorescent lineage tracking to examine the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor exhibited multicolor fluorescence, showing that it contains multiclonal cancer cell populations. Peritoneal cytology and in situ fluorescent imaging of micrometastases revealed that multicolored cancer cell clusters formed within the peritoneal cavity and collectively attached to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients elicited cancer cell clustering, which was inhibited by anticoagulants. Immunofluorescence and immunoblotting revealed that cancer cell clusters formed in vitro and in vivo were associated with fibrin formation. Moreover, tissue factor knockout in cancer cells significantly reduced fibrin formation, cell clustering, peritoneal attachment, and peritoneal metastasis. In an in vitro adhesion assay, fibrin formation facilitated the cancer cell-mesothelial cell adhesion. Thus, these results demonstrate that cancer cells activate the coagulation cascade through tissue factor to form fibrin-mediated cell clusters and accelerate peritoneal attachment, leading to the development of polyclonal peritoneal metastasis. Our findings identify a novel mechanism of peritoneal metastasis that may be a potential therapeutic target. Citation Format: Makoto Miyazaki, Yuko Nagamura, Hideki Yamaguchi. Tissue factor-induced fibrinogenesis and cancer cell clustering contribute to polyclonal peritoneal metastasis in diffuse-type gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2473.

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA–mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP–dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion. [Cancer Res 2009;69(22):8594–602]

Neuroblastomas harbor mutations in the nonreceptor anaplastic lymphoma kinase (ALK) in 8% to 9% of cases where they serve as oncogenic drivers. Strategies to reduce ALK activity offer clinical interest based on initial findings with ALK kinase inhibitors. In this study, we characterized phosphotyrosine-containing proteins associated with ALK to gain mechanistic insights in this setting. Flotillin-1 (FLOT1), a plasma membrane protein involved in endocytosis, was identified as a binding partner of ALK. RNAi-mediated attenuation of FLOT1 expression in neuroblastoma cells caused ALK dissociation from endosomes along with membrane accumulation of ALK, thereby triggering activation of ALK and downstream effector signals. These features enhanced the malignant properties of neuroblastoma cells in vitro and in vivo. Conversely, oncogenic ALK mutants showed less binding affinity to FLOT1 than wild-type ALK. Clinically, lower expression levels of FLOT1 were documented in highly malignant subgroups of human neuroblastoma specimens. Taken together, our findings suggest that attenuation of FLOT1-ALK binding drives malignant phenotypes of neuroblastoma by activating ALK signaling. Cancer Res; 74(14); 3790–801. ©2014 AACR.

Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. To date, most studies of N-WASP have focused on intracellular and morphological phenomena, such as vesicle transport and filopodium formation. We investigated the importance of N-WASP in epithelial morphogenesis, using Madin-Darby canine kidney epithelial cells, which form branching tubules when cultured with hepatocyte growth factor (HGF) in collagen gel. We established MDCK cell lines that overexpress wild-type N-WASP (WT-NW) or a dominant-negative form of N-WASP (DN-NW). WT-NW and parental Madin-Darby canine kidney cells formed branching tubules in collagen gel in response to HGF. However, formation of branching tubules was suppressed in DN-NW cells. During tubulogenesis, endogenous N-WASP accumulated at cell extensions protruding from the walls of the cysts and at the tips of the extending tubules. Gross cell morphology, cell-cell adhesion, cell polarity, and scattering in response to HGF were unaffected in WT-NW and DN-NW cells. In contrast, directed cell migration and HGF-induced invasion were significantly repressed in DN-NW cells. These results indicate that N-WASP regulates HGF-induced cell migration and invasion, which are required for epithelial tubulogenesis.

Scirrhous gastric carcinoma (SGC; also called diffusely infiltrative carcinoma, Borrmann's type-IV carcinoma, or the linitis plastica-type carcinoma) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC. Citation Format: Hideki Yamaguchi, Kazuyoshi Yanagihara, Masakazu Yashiro, Ryuichi Sakai. Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3601. doi:10.1158/1538-7445.AM2014-3601

Invadopodia are ventral membrane protrusions that are formed by invasive cancer cells cultured on physiological substrates. They are actin-based structures and promote the degradation of the extracellular matrix by focally localizing matrix metalloproteinases. Accumulating evidence suggests that invadopodia play an important role in local invasion and distant metastasis of malignant tumors. Actinin is an actin-binding protein that forms dimers and crosslinks actin filaments. There are four isoforms of actinin in mammals and they are divided into the muscle type (actinin-2 and -3) and the non-muscle type (actinin-1 and -4). Actinin-1 is ubiquitously expressed and involved in the formation of focal adhesions, while actinin-4 is strongly expressed at the invading front of advanced cancers and its expression is correlated with poor prognosis. Although actinin-4 is thought to regulate cell motility and invasion, the detailed molecular mechanisms remain unclear. Moreover, functional differences between the two isoforms have yet to be fully defined. In this study, we studied the roles of and functional differences between actinin-1 and -4 in invadopodia formation to determine the impact of actinin-4 expression on cancer cells. Immunofluorescence analysis showed that both actinin-1 and -4 clearly localized at invadopodia in RPMI-7951 human melanoma cells and there was no obvious difference in their localization patterns. Likewise, invadopodia formation and degradation of fluorescent gelatin matrix were blocked by siRNA-mediated knockdown of either actinin-1 or -4. In contrast, only actinin-4, but not actinin-1, promoted invadopodia-mediated gelatin degradation activity when overexpressed in RPMI7951 cells. Similar results were obtained when MDA-MB-231 human breast cancer cells and SCC61 human head and neck squamous cell carcinoma cells were analyzed in the same manner. These results indicate that both actinin-1 and -4 are essential components of invadopodia, but only actinin-4 possesses an ability to promote invadopodia formation. Therefore, further analysis is conducted now to identify the unique functions actinin-4 in invadopodia formation. Citation Format: Yuumi Ito, Hideki Yamaguchi, Kiyoko Fukami, Kazufumi Honda, Nami Miura, Tesshi Yamada, Ryuichi Sakai. Functional differences of actinin isoforms in the formation of invadopodia by invasive cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4070. doi:10.1158/1538-7445.AM2014-4070

Degradation of extracellular matrix that is present in the basement membrane and tumor stroma is essential for local invasion and formation of metastatic sites by malignant cancer cells. Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that have been shown to function in cancer invasion and metastasis. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain poorly understood. The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate phosphoinositides at the D-3 position of the inositol headgroup. PI3Ks mediate the signal transduction of extracellular stimuli and regulate diverse cellular events. Uncontrolled activation of the PI3K signaling pathway leads to tumorigenesis and tumor malignancy. In this study, we investigated the role of PI3K signaling during invadopodia formation. We found that in human breast cancer cells both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells. Citation Format: Hideki Yamaguchi, Nachi Yoshida, Ryuichi Sakai. The role of PI3-kinase signaling pathway in invadopodia formation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A82.

Scirrhous gastric carcinoma (SGC; also called diffusely infiltrative carcinoma, Borrmann's type-IV carcinoma, or the linitis plastica-type carcinoma) have the worst prognosis among various types of gastric cancer, owing to highly progressive invasion, frequent metastasis to lymph nodes, and high incidence of peritoneal dissemination. SGC cells rapidly and diffusively infiltrate through the submucosa without forming obvious tumor mass and induce extensive stromal fibrosis, resulting in thickening and stiffening of the gastric wall. Because histophathological analyses have shown that massive proliferation of stromal fibroblasts occur within lesions of SGC, cancer-associated fibroblasts (CAFs) have been proposed to support the progression of SGC. However, biological and molecular basis of the interaction between SGC cells and CAFs remains largely unknown. In this study, we investigated the roles of CAFs in invasion and extracellular matrix (ECM) remodeling by SGC cells. When 44As3 SGC cells were co-cultured with CaF37 SGC-derived fibroblasts on 3D Matrigel, they formed large cell aggregates that invade into the Matrigel. Other SGC cell lines, HSC59 and HSC44PE, but not differentiated non-SGC gastric caner cell lines, MKN7, MKN1, and MKN74, formed invasive cell aggregate with CaF37 cells. Time-lapse imaging of SGC and CaF37 cells, and of fluorescent microsphere embedded in the Matrigel revealed that this process is associated with extensive contraction and remodeling of ECM. In order to further characterize the ECM remodeling activity of CAFs, we cultured 44As3 and CaF37 cells on fluorescent gelatin-coated coverslips. CaF37 cells caused disruption of the gelatin matrix and this activity was further promoted by co-culturing them with 44As3 cells. Immunoblotting and immnofluorescence analyses revealed that phosphorylation of myosin light chain was significantly increased in CaF37 cells co-cultured with 44As3 cells. Finally, a myosin II inhibitor blebbistatin blocked the three-dimensional invasion and ECM remodeling by 44As3 and CaF37 cells. These results indicate that SGC cells promote actomyosin-mediated contractility of stromal fibroblasts to remodel ECM during invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 47. doi:1538-7445.AM2012-47

The prognosis of patients with scirrhous gastric carcinoma (SGC) has been very poor. SGC cells do not form obvious tumor mass, but cause diffuse infiltration and eventually rigid thickening of the gastric wall. Furthermore, SGC cells frequently infiltrate through gastric serosa and cause peritoneal dissemination. However, the mechanisms underlying the progression of SGC have not yet been fully elucidated. In the present study, we performed immunoblotting and proteomic analyses of tyrosine-phosphorylated proteins in differentiated non-SGC cell lines (MKN7, MKN1, and MKN74) and poorly differentiated or SGC cell lines (MKN45, NUGC-4, KATO III, HSC59, 58As9, HSC44-PE, and 44As3) to identify signaling pathways and molecules that are characteristics of SGC. We found that the tyrosine kinase receptor c-Met is overexpressed and highly phosphorylated in SGC cell lines. c-Met is a cell surface receptor for HGF and implicated in the growth, survival, invasion, and metastasis of various human cancers. Amplification of c-Met gene in SGC was also previously reported. We analyzed the downstream signaling pathways of c-Met in SGC cells by using two c-Met inhibitors PHA-665752 and JNJ-38877605. Treatment of 58As9 cells with the c-Met inhibitors markedly inhibited phosphorylation of Erk, Akt, and Stat3, but promoted phosphorylation of Src. In contrast, 44As3 cells treated with the c-Met inhibitors showed no significant changes in phosphorylation of these molecules, in spite of successful inhibition of c-Met phosphorylation. Reflecting these observations, c-Met inhibitors blocked cell proliferation and caused morphological alterations in 58As9 cells but not in 44As3 cells. Similar results were obtained when MKN45 and NUGC-4 cells were analyzed in the same manner: MKN45 cells but not NUGC-4 cells were sensitive to c-Met inhibition. These results suggest that SGC can be classified into two subtypes according to their dependencies on the c-Met signaling pathway. Further studies are now conducted to identify therapeutic targets in c-Met-independent SGC cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1244. doi:1538-7445.AM2012-1244

Degradation of extracellular matrix (ECM) that is present in the basement membrane and tumor stroma is essential for local invasion and formation of metastatic sites by malignant cancer cells. Invadopodia are ECM-degrading protrusions formed by a variety of invasive cancer cell types and are thought to function in cancer invasion and metastasis. Many components of invadopodia, such as proteins involved in actin polymerization, cell signaling, and ECM degradation, have been reported to date. However, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that produce D-3 phosphoinositides. PI3Ks mediate the signal transduction of extracellular stimuli and regulate diverse cellular events. Uncontrolled activation of the PI3K signaling pathway leads to several pathological phenomena, including tumorigenesis and tumor malignancy. We investigated the role of PI3K signaling during invadopodia formation. We found that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix was blocked by treatment with PI3K inhibitors or sequestration of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. Mammals have 8 PI3K enzymes that are subdivided into three general classes (I-III) on the basis of their domain structures and substrate specificities. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α is selectively involved in invadopodia formation. p110α is encoded by the PIK3CA gene, which is one of the most frequently amplified and mutated genes identified in human cancers. Clinical studies revealed that activating mutations of PIK3CA are associated with the development of invasive and metastatic phenotypes and poor patient prognosis. The expression of p110α with the activating mutations promoted invadopodia-mediated invasive activity. Furthermore, knocking down PDK1 and Akt, the downstream effectors of PI3K signaling, suppressed invadopodia formation induced by the p110α mutants. These data suggest that the PI3K signaling pathway via p110α regulates invadopodia-mediated invasion of breast cancer cells. Targeting p110α may be a potential therapeutic approach for controlling invasion of malignant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2011-4748