Ryo Yuge

mTOR and PDGF pathway blockade inhibits liver metastasis of colorectal cancer by modulating the tumor microenvironment.

Tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells, and platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations. Activation of PI3K-AKT-mTOR signaling is frequently observed in many cancer types. We investigated whether the mTOR inhibitor everolimus, alone or in combination with the PDGF-R tyrosine kinase inhibitor nilotinib, can inhibit growth and metastasis of human colon cancer. The effects of nilotinib and everolimus on tumor growth and metastasis were examined in an orthotopic mouse model of human colon cancer and a model of liver metastasis. After treatment with nilotinib (versus distilled water), the stromal reaction of xenografts growing in the cecal wall and liver was significantly decreased. After treatment with everolimus, the stromal reaction did not decrease, but tumor cell proliferation and microvessel density decreased. With the two drugs in combination, both stromal reaction and tumor cell proliferation decreased and apoptosis of tumor cells increased, resulting in remarkable inhibition of tumor growth at both the orthotopic and the metastatic site. Concurrent inhibition of tumor cells and activated stromal cells by a PDGF-R tyrosine kinase inhibitor and an mTOR inhibitor used in combination may represent a novel therapeutic approach for colorectal cancer.

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma.

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma‐associated fibroblasts (CAFs) expressed platelet‐derived growth factor receptor‐β (PDGFR‐β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow‐derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1–3, TIE2, PDGFR‐β, and fibroblast growth factors) and tumor cells (c‐KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor‐promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co‐implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor‐inhibitory effect of regorafenib was more obvious in tumors developed by co‐implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell–MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact

We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow–derived mesenchymal stem cells (MSCs) migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT)–related genes such as fibronectin (FN), SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.

KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30–40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apcflox/flox; LSL-KrasG12D and CDX2P-G22Cre;Apcflox/flox mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription–PCR (qRT–PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT–PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin–nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin–NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Regression of Cecal MALT Lymphoma after Antibiotic Treatment in a Patient with Helicobacter pylori Infection.

A 63-year-old man with abdominal discomfort was referred to our hospital. Colonoscopy revealed a hemispherical-shaped protruding cecal mass of approximately 10 mm in size with a normal mucosal surface. Biopsy specimens showed nodules consisting of the proliferation of atypical lymphoid cells. Mucosa-associated lymphoid tissue (MALT) lymphoma was diagnosed based on the histological and immunohistochemical findings. Since upper gastrointestinal endoscopy demonstrated Helicobacter pylori-associated atrophic gastritis, eradication therapy was administered. The cecal mass disappeared completely within three months after triple therapy. Therefore, H. pylori eradication therapy may be a useful treatment option for cecal MALT lymphoma.

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.

Abstract 5068: Treatment with regorafenib inhibits the tumor-promoting effect of bone marrow-derived mesenchymal stem cells in an orthotopic nude mice model of colon cancer

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to tumor stroma as well as injured tissue. We reported that, in an orthotopic nude mice model of colon cancer, MSCs travel to tumor stroma and differentiate into carcinoma-associated fibroblasts (CAFs), and then, they promote growth and metastasis of colon cancer. We also found that CAFs express platelet-derived growth factor receptor-beta (PDGFR-β), at a high level and that imatinib therapy targeting PDGFR in CAFs combined with administration of a cytotoxic drug significantly inhibits growth and metastasis of human colon cancer. A novel oral multi-kinase inhibitor, regorafenib not only inhibits oncogenic receptor tyrosine kinases such as c-KIT, RET, and B-RAF, but also inhibits stromal cells which express VEGFR1-3, TIE2, PDGFR-β, and fibroblast growth factor receptor 1 (FGFR1). These cell surface receptor tyrosine kinases are known to be involved in tumor neovascularization, vessel stabilization, lymphatic vessel formation as well as activation of stroma. We examined whether the effect of MSCs on tumor growth and metastasis was inhibited by regorafenib. KM12SM human colon cancer cells alone or KM12SM cells mixed with MSCs in a 1:2 ratio were transplanted into the cecal wall of nude mice. Orthotopic transplantation of KM12SM cells + MSCs, in comparison to transplantation of KM12SM cells alone, resulted in tumors of greater weight and volume, and higher rate of lymphatic metastasis. Co-injection of MSCs with tumor cells promoted tumor growth and metastasis of colon cancer by enhancing angiogenesis, lymphangiogenesis, and tumor cell proliferation and by inhibiting tumor cell apoptosis. When CRC tumor + MSCs bearing animals were treated with regorafenib (by oral gavage once daily at 10 mg/kg for 35 days), primary tumor size or the number of lymph node metastases were significantly decreased compared to those treated with vehicle, and reached to the level with no significant difference from KM12SM tumor cells alone. Our data suggest that targeting tumor microenvironmental signaling pathways by regorafenib influences the interaction between bone marrow-derived MSCs and tumor cells and, hence, inhibits the progressive growth of colon cancer. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Ryo Yuge, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Treatment with regorafenib inhibits the tumor-promoting effect of bone marrow-derived mesenchymal stem cells in an orthotopic nude mice model of colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5068. doi:10.1158/1538-7445.AM2015-5068

Abstract 1987: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces gastric cancer cell motility and metastasis

Accumulating evidences suggest that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), play a role in cancer progression by interaction with their surrounding collagen matrix. Although abnormal expression of DDRs is reported in some human cancers, little is known about the expression and function of DDRs in gastric cancer progression. In this study, we investigated expression and the role of DDR1 in the tumor progression of human gastric cancer. The expression and distribution of DDR1 was analyzed by immunohistochemistry in 127 human gastric cancer cases, and the expression level of DDR1 in 7 human gastric cancer cell lines was analyzed by RT-PCR and western blotting. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1 silenced gastric cancer cells. The effect of DDR1 on the tumor growth and metastasis were examined in orthotopically implanted nude mice model and a model of liver metastasis. DDR1 was expressed in human gastric cancer cell lines, and expression of DDR1 in human gastric tumor was associated with poor prognosis. Among 7 gastric cancer cell lines, MKN74 expressed DDR1 at highest level. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumor significantly decreased angiogenesis and lymphangiogensesis, thereby leading to reduction of lymph node metastasis and liver metastasis. We showed that treatment of DDR1 inhibitor also inhibited lymph node metastasis in an orthotopic nude mouse model These results suggest that DDR1 is involved in GC tumor progression and metastasis. DDR1 could be not only an independent prognostic classifier, but also a promising therapeutic target in patients with GC. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Hidehiko Takigawa, Shinji Tanaka, Kazuaki Chayama, Wataru Yasui. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces gastric cancer cell motility and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2017-1987

Combination therapy using molecular-targeted drugs modulates tumor microenvironment and impairs tumor growth in renal cell carcinoma

Tumor growth and metastasis are determined not by cancer cells alone but also by a variety of stromal cells, various populations of which overexpress platelet‐derived growth factor receptors (PDGF‐Rs). In addition, activation of PI3K‐AKT‐mammalian target of rapamycin (mTOR) signaling is frequently observed in many cancer types as well. mTOR comprises a serine/threonine kinase that increases the production of proteins that stimulate key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis. In this study, we investigated the impact of molecular‐targeting agents including PDGF‐R and mTOR inhibitors on the tumor stroma of human kidney cancer and examined the efficacy of combination therapy with these agents against this disease. Treatment with sunitinib did not suppress tumor growth, but significantly decreased stromal reactivity, microvessel density, and pericyte coverage of tumor microvessels in an orthotopic mouse model. In contrast, treatment with everolimus decreased tumor growth and microvessel density but not stromal reactivity. However, sunitinib and everolimus in combination reduced both the growth rate and stromal reaction. These findings suggest that target molecule‐based inhibition of the cancer–stromal cell interaction appears promising as an effective antitumor therapy.

Abstract 5070: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by regulating the interactions of tumor cells with their surrounding collagen matrix. We investigated the role of DDR1 in the tumor progression of colorectal cancer. We examined the DDR1 mRNA and protein expression levels in a series of human colorectal cancer cell lines by RT-PCR and Western blotting. We also analyzed the DDR1 protein expression in 27 surgical specimens of colorectal cancer by immunohistochemistry. Migration, invasion, and proliferation assays were conducted with a high-content time-lapse assay system in a series of colon cancer cell lines. Nilotinib, a well-known DDR1 inhibitor, and DDR1-silenced colon cancer cells were used for inhibition of DDR1 tyrosine kinase activity. In an in vivo study, we orthotopically implanted KM12SM human colon cancer cells into the cecum of nude mice and administered nilotinib (100 mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically. DDR1 mRNA and protein expression was observed in the human colon cancer cell lines and in cancer cells of most of the surgical specimens. Colon cancer cell lines KM12SM, KM12c, LoVo, and HT29 exposed to nilotinib (20 μM) showed reduced migration. When incubated with type I collagen, however, these cells showed increased migratory activity. Nilotinib treatment significantly inhibited this effect. The migratory activity of DDR1-silenced cells was not affected by nilotinib treatment. The orthotopic xenografts grew invasively as aggressive tumors with a stromal reaction, but under nilotinib treatment, the stromal reaction was reduced, and tumors grew expansively. In addition, the numbers of lymph node metastases were significantly reduced. Disruption of DDR1 attenuated colon cancer cell migration and led to altered primary tumor morphology and to impaired lymph node metastasis. DDR1 may be a promising therapeutic target in patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2015-5070