Kazuo Yasumoto

Role of the CXCL12/CXCR4 axis in Peritoneal Carcinomatosis of Gastric Cancer

Peritoneal carcinomatosis is a frequent cause of death in patients with advanced gastric carcinoma. Because chemokines are now considered to play an important role in the metastasis of various malignancies, we hypothesized that they may be involved in the development of peritoneal carcinomatosis by gastric carcinoma. Human gastric carcinoma cell lines, which were all highly efficient in generating malignant ascites in nude mice upon i.p. inoculation, selectively expressed CXCR4 mRNA and protein. In particular, NUGC4 cells expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its ligand CXCL12. CXCL12 enhanced proliferation and rapid increases in phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase of NUGC4 cells. We also showed that AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor growth and ascitic fluid formation in nude mice inoculated with NUGC4 cells. Additionally, we examined human clinical samples. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high concentrations of CXCL12 (4.67 ng/mL). Moreover, immunohistochemical analysis showed that 22 of 33 primary gastric tumors with peritoneal metastasis were positive for CXCR4 expression (67%), whereas only 4 of 16 with other distant metastasis were positive (25%). Notably, 22 of 26 CXCR4-expressing primary tumors developed peritoneal metastases (85%). CXCR4 positivity of primary gastric carcinomas significantly correlated with the development of peritoneal carcinomatosis (P < 0.001). Collectively, our results strongly suggest that the CXCR4/CXC12 axis plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. Thus, CXCR4 may be a potential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

Chemokine receptors in cancer metastasis and cancer cell‐derived chemokines in host immune response

The chemotactic cytokines called chemokines are a superfamily of small secreted cytokines that were initially characterized through their ability to prompt the migration of leukocytes. Attention has been focused on the chemokine receptors expressed on cancer cells because cancer cell migration and metastasis show similarities to leukocyte trafficking. CXC chemokine receptor 4 (CXCR4) was first investigated as a chemokine receptor that is associated with lung metastasis of breast cancers. Recently, CXCR4 was reported to be a key molecule in the formation of peritoneal carcinomatosis in gastric cancer. In the present review, we highlight current knowledge about the role of CXCR4 in cancer metastases. In contrast to chemokine receptors expressed on cancer cells, little is known about the roles of cancer cell‐derived chemokines. Cancer tissue consists of both cancer cells and various stromal cells, and leukocytes that infiltrate into cancer are of particular importance in cancer progression. Although colorectal cancer invasion is regulated by the chemokine CCL9‐induced infiltration of immature myeloid cells into cancer, high‐level expression of cancer cell‐derived chemokine CXCL16 increases infiltrating CD8+ and CD4+ T cells into cancer tissues, and correlates with a good prognosis. We discuss the conflicting biological effects of cancer cell‐derived chemokines on cancer progression, using CCL9 and CXCL16 as examples. (Cancer Sci 2007; 98: 1652–1658)

Properties of Pro-Inflammatory Cell Type-Specific Leukocyte Chemotactic Cytokines, Interleukin 8(IL-8) and Monocyte Chemotactic and Activating Factor (MCAF)

Inflammation is a host defensive reaction to tissue injuries caused by various conditions such as trauma, infections, invasion of foreign particles, ischemia-reperfusion syndrome, malignant tumors, and autoimmune diseases. The early reactions of inflammations consist of plasma leakage and leukocyte infiltration into the lesion. The types of infiltrating leukocytes depend upon the kind, degree, and timing of tissue injuries. Although several factors such as C5a, leukotriene B4 and bacteriaderived formyl peptides have been long known as leukocyte chemotactic factors, these factors chemoattract any kind of leukocytes, thus suggesting the existence of cell type-specific leukocyte chemotactic factors (28). Over the past several years, numerous members of a new family of heparinbinding polypeptide cytokines with a molecular weight of 8-10 kDa have been identified (Ref. 25) (Table 1) . These factors have 4 cysteine residues at the wellconserved positions and are presumed to possess a similar structure (25). These cytokines are produced massively and rapidly by various kinds of cells in response to inflammatory stimuli including lipopolysaccharide (LPS) , viruses, interleukin 1 (IL-1), tumor necrosis factor (TNF) and so on [Tables 2 and 3 show the cell sources and inducers of interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF) described below]. Furthermore, it has become evident that several of these cytokines have chemotactic activity for specific type(s) of leukocytes, suggesting that these factors are bona fide chemoattractants involved in the leukocyte recruitment seen in the inflammatory processes. These factors are subdivided into two subgroups depending on whether the first two cysteines are separated by a single amino acid or in an adjacent position (Table 1). The former are called intercrine

Inhibition of GSK‐3β activity attenuates proliferation of human colon cancer cells in rodents

The authors’ recent discovery that glycogen synthase kinase‐3β (GSK‐3β) participates in colon cancer cells’ survival and proliferation prompted us to investigate whether GSK‐3β inhibition alters proliferation of colon cancer cells in vivo. Groups of four or five athymic mice (Balb/c, nu/nu) with subcutaneous xenografts of SW480 human colon cancer cells were treated with dimethyl sulfoxide (DMSO) or different doses (1, 2 and 5 mg/kg body weight) of either small‐molecule GSK‐3β inhibitor (SB‐216763 and AR‐A014418) by intraperitoneal injection three times per week for 5 weeks. Compared with DMSO (a diluent of the GSK‐3β inhibitors) as a control, either GSK‐3β inhibitor significantly inhibited proliferation of cancer cell xenografts in the rodents in a dose‐dependent manner. Histochemical and immunohistochemical analysis of tumor xenografts demonstrated a significant, dose‐dependent decrease in fractions of proliferating cells and an increase in the incidence of apoptosis of cancer cells in mice treated with either GSK‐3β inhibitor. No adverse events or effects were observed in the rodents during the course of treatment, except for rare lethal accidents due to intraperitoneal injection. Morphological examination showed no apparent pathologic changes in major organs including the lungs, liver, pancreas, kidneys, spleen and large bowel of rodents treated with DMSO and the GSK‐3β inhibitors. The results indicate that the GSK‐3β inhibitors would be a novel class of therapeutic agent for colon cancer. (Cancer Sci 2007; 98: 1388–1393)

Human MCAF Gene Transfer Enhances the Metastatic Capacity of a Mouse Cachectic Adenocarcinoma Cell Line in Vivo

AbstractPurpose. To evaluate the effect of monocyte chemotactic and activating factor (MCAF/MCP-1/JE) on tumor progression and metastasis. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCAF expression vector. Spontaneous lung metastases were determined in mouse. Results. The production of MCAF reached 0.4 ng/ml in vitro when transfectant cells were cultured at a cell density of 5 × 104 cells/ml for 3 days. Transfection of MCAF expression vector did not affect the growth rate in vitro. Also, after MCAF-transfection, the size of tumors after intra-footpad inoculation was similar to that of the parental cells. When the primary tumors were resected on the 10th day after inoculation, the incidence of spontaneous lung metastasis was less than 20% in both cells. The number of endothelial cells in the primary tumor rapidly increased from the 10th to the 14th day after inoculation, as revealed by immunohistochemical staining. In accordance with enhanced angiogenesis, the incidence rates of spontaneous metastasis increased when the primary tumors were resected on the 14th day after inoculation. Moreover, the spontaneous lung metastases were augmented in the animals injected with MCAF-transfectants compared to those injected with parental cells with a concomitant increase of angiogenesis. Conclusions. These results suggest that MCAF may augment the metastatic potential by modulating tumor associated angiogenesis.

The EGFR Ligands Amphiregulin and Heparin-Binding EGF-like Growth Factor Promote Peritoneal Carcinomatosis in CXCR4-Expressing Gastric Cancer

Purpose: Peritoneal carcinomatosis, often associated with malignant ascites, is the most frequent cause of death in patients with advanced gastric cancer. We previously showed that the CXCR4/CXCL12 axis is involved in the development of peritoneal carcinomatosis from gastric cancer. Here, we investigated whether epidermal growth factor receptor (EGFR) ligands are also involved in the development of peritoneal carcinomatosis from gastric cancer. Experimental Design: The functional involvement of expression of the ErbB family of receptors and/or EGFR ligands was examined in CXCR4-expressing human gastric cancer cells and fibroblasts, clinical samples (primary tumors and ascites), and an animal model. Results: High concentration of the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF), as well as of CXCL12, were present in malignant ascites. Human gastric cancer cell lines and primary gastric tumors, with high potential to generate peritoneal carcinomatosis, expressed high levels of EGFR and CXCR4 mRNA and protein. Both amphiregulin and HB-EGF enhanced the proliferation, migration, and functional CXCR4 expression in highly CXCR4-expressing gastric cancer NUGC4 cells. Amphiregulin strongly enhanced the proliferation of NUGC4 cells, whereas HB-EGF markedly induced the migration of fibroblasts. Moreover, HB-EGF and CXCL12 together enhanced TNFα-converting enzyme (TACE)-dependent amphiregulin shedding from NUGC4 cells. In an experimental peritoneal carcinomatosis model in mice, cetuximab effectively reduced tumor growth and ascites formation. Conclusions: Our results strongly suggest that the EGFR ligands amphiregulin and HB-EGF play an important role, interacting with the CXCL12/CXCR4 axis, in the development of peritoneal carcinomatosis from gastric cancer, indicating that these two axes may be potential therapeutic targets for peritoneal carcinomatosis of gastric carcinoma. Clin Cancer Res; 17(11); 3619–30. ©2011 AACR.

Pleural Mesothelioma Instigates Tumor-Associated Fibroblasts To Promote Progression via a Malignant Cytokine Network

The tumor microenvironment is crucial to the progression of various malignancies. Malignant pleural mesothelioma (MPM), which originates from the pleura, grows aggressively in the thoracic cavity. Here we describe an orthotopic implantation SCID mouse model of MPM and demonstrate that α-SMA-positive fibroblast-like cells accumulate in the tumors produced by the human MPM cell lines MSTO-211H and Y-Meso-14. We assessed the interaction between MPM cells and their microenvironments, focusing on tumor-associated fibroblasts. MSTO-211H and Y-Meso-14 cells produced fibroblast growth factor-2 (FGF-2) and/or platelet-derived growth factor-AA (PDGF-AA); they also enhanced growth, migration, and production of hepatocyte growth factor (HGF) by human lung fibroblast MRC-5 cells. MRC-5 cells stimulated HGF-mediated growth and migration of MSTO-211H and Y-Meso-14 cells in an in vitro coculture system. In the orthotopic model, tumor formation by MSTO-211H and Y-Meso-14 cells was significantly inhibited by TSU-68, an inhibitor of FGF, VEGF, and PDGF receptors; imatinib, an inhibitor of PDGF receptors; and NK4, an antagonist of HGF. Histological analyses of clinical specimens from 51 MPM patients revealed considerable tumor-associated fibroblasts infiltration and expression of HGF, together with FGF-2 or PDGF-AA, in tumors. These findings indicate that MPM instigates tumor-associated fibroblasts, promoting tumor progression via a malignant cytokine network. Regulation of this cytokine network may be therapeutically useful for controlling MPM.

Ability of the Met kinase inhibitor crizotinib and new generation EGFR inhibitors to overcome resistance to EGFR inhibitors

Purpose Although EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have shown dramatic effects against EGFR mutant lung cancer, patients ultimately develop resistance by multiple mechanisms. We therefore assessed the ability of combined treatment with the Met inhibitor crizotinib and new generation EGFR-TKIs to overcome resistance to first-generation EGFR-TKIs. Experimental Design Lung cancer cell lines made resistant to EGFR-TKIs by the gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression and mice with tumors induced by these cells were treated with crizotinib and a new generation EGFR-TKI. Results The new generation EGFR-TKI inhibited the growth of lung cancer cells containing the gatekeeper EGFR-T790M mutation, but did not inhibit the growth of cells with Met amplification or HGF overexpression. In contrast, combined therapy with crizotinib plus afatinib or WZ4002 was effective against all three types of cells, inhibiting EGFR and Met phosphorylation and their downstream molecules. Crizotinib combined with afatinib or WZ4002 potently inhibited the growth of mouse tumors induced by these lung cancer cell lines. However, the combination of high dose crizotinib and afatinib, but not WZ4002, triggered severe adverse events. Conclusions Our results suggest that the dual blockade of mutant EGFR and Met by crizotinib and a new generation EGFR-TKI may be promising for overcoming resistance to reversible EGFR-TKIs but careful assessment is warranted clinically.

mTOR Inhibitors Control the Growth of EGFR Mutant Lung Cancer Even after Acquiring Resistance by HGF

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is a critical problem in the treatment of EGFR mutant lung cancer. Several mechanisms, including bypass signaling by hepatocyte growth factor (HGF)-triggered Met activation, are implicated as mediators of resistance. The mammalian target of rapamycin (mTOR), is a downstream conduit of EGFR and MET signaling, and is thus considered a therapeutically attractive target in the treatment of various types of cancers. The purpose of this study was to examine whether 2 clinically approved mTOR inhibitors, temsirolimus and everolimus, overcome HGF-dependent resistance to EGFR-TKIs in EGFR mutant lung cancer cells. Both temsirolimus and everolimus inhibited the phosphorylation of p70S6K and 4E-BP1, which are downstream targets of the mTOR pathway, and reduced the viability of EGFR mutant lung cancer cells, PC-9, and HCC827, even in the presence of HGF in vitro. In a xenograft model, temsirolimus suppressed the growth of PC-9 cells overexpressing the HGF-gene; this was associated with suppression of the mTOR signaling pathway and tumor angiogenesis. In contrast, erlotinib did not suppress this signaling pathway or tumor growth. Multiple mechanisms, including the inhibition of vascular endothelial growth factor production by tumor cells and suppression of endothelial cell viability, contribute to the anti-angiogenic effect of temsirolimus. These findings indicate that mTOR inhibitors may be useful for controlling HGF-triggered EGFR-TKI resistance in EGFR mutant lung cancer, and they provide the rationale for clinical trials of mTOR inhibitors in patients stratified by EGFR mutation and HGF expression status.

Triple inhibition of EGFR, Met, and VEGF suppresses regrowth of HGF-triggered, erlotinib-resistant lung cancer harboring an EGFR mutation.

Introduction: Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer. Methods: Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene–transfected PC-9 (PC-9/HGF) cells were examined. Results: Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment. Conclusion: These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis.