Mieko Onoyama

Stroma‐directed imatinib therapy impairs the tumor‐promoting effect of bone marrow‐derived mesenchymal stem cells in an orthotopic transplantation model of colon cancer

Bone marrow‐derived mesenchymal stem cells (MSCs) are reported to contribute to formation of tumor‐promoting stromal cells. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma‐associated fibroblast (CAF)‐like cells. We also found that CAFs express platelet‐derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor‐promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. Orthotopic transplantation and splenic injection of human MSCs along with KM12SM human colon cancer cells, in comparison with transplantation of KM12SM cells alone, resulted in significantly greater promotion of tumor growth and liver metastasis. The KM12SM + MSC xenograft enhanced cell proliferation and angiogenesis and inhibited tumor cell apoptosis. When tumor‐bearing animals were treated with imatinib, there was no significant increase in primary tumor volume or total volume of liver metastases, despite the KM12SM+MSC xenograft, and survival in the mixed‐cell group was prolonged by imatinib treatment. Moreover, the ability of MSCs to migrate to tumor stroma was impaired, and the number of MSCs surviving in the tumor microenvironment was significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration of MSCs. Our data suggest that blockade of PDGF signaling pathways influences the interaction between bone marrow‐derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.

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.

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 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

Abstract 146: Combining molecular targeted drugs to inhibit both cancer cells and activated stromal cells in colon cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Recent studies have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. We have shown previously that platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations, but not by cancer cells themselves in human colon cancer tissues. Blockade of PDGF-R signaling pathways in tumor-associated stromal cells has been shown to inhibit the stromal reaction. Activation of phosphoinositide 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling is frequently observed in many cancer types and has been implicated in the pathogenesis of various diseases. mTOR is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell proliferation, metabolism, survival, and angiogenesis. However, the effects of mTOR inhibitors on the stromal component of tumor tissues have not been studied. In the present study, the effects of nilotinib (PDGF-R tyrosine kinase inhibitor) and everolimus (mTOR inhibitor) on tumor development and metastasis, were examined using orthotopic and experimental liver metastatic models of human colon cancer, whereby KM12SM cells were implanted into the cecum and spleen of nude mice, respectively. Groups of mice received saline (control), nilotinib (100mg/kg/day) or everolimus (1mg/kg/day), or a combination of nilotinib (100mg/kg/day) and everolimus (1mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically and immunohistochemically for comparison between the treatment group and control group. After treatment with nilotinib, the stromal reaction was significantly decreased. After treatment with everolimus, the stromal reaction was not decreased, but tumor cell proliferation and microvessel density were decreased. With the combination therapy, both tumor cell proliferation and the stromal reaction were decreased and apoptosis of tumor cells was increased, resulting in a remarkable decrease in tumor size and the number of metastatic liver lesions. Therefore, concurrent inhibition of tumor cells and activated stromal cells by combination therapy may represent a novel molecular-targeted approach for patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Yuichiro Tanaka, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Combining molecular targeted drugs to inhibit both cancer cells and activated stromal cells in colon cancer. [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 146. doi:10.1158/1538-7445.AM2014-146

Abstract 180: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of fibronectin (FN) mRNA was increased markedly by direct co-culture of KM12SM cells with MSCs. FN, a glycoprotein in extracellular matrix and also a maker of epithelial-mesenchymal transition (EMT), has been implicated in the development of several types of human cancer. Many reports indicate that EMT is an important step during the cascade of tumor progression. However, the potential role of FN in carcinogenesis is not fully elucidated. We examined FN expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 87 tumors were analyzed. Levels of FN expression in tumor cells correlated with disease stage, and FN immunoreactivity was detected in invasive cancers, especially at the invasive edge, but less in mucosal cancers or adenomas. Furthermore, in an orthotopic nude mouse model of colon cancer, mRNA expression levels of FN were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone.These findings suggest that the interaction between cancer cells and MSCs recruited into tumor stroma may play a role in tumor growth and metastasis via induction of FN expression in invasive colon cancer. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Yuichiro Tanaka, Ryo Yuge, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon 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 180. doi:10.1158/1538-7445.AM2014-180

Abstract 2074: Blockade mTOR and PDGF pathways by combination of molecular-targeted drugs inhibited cancer-stromal cell interaction in colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. We have shown previously that platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations, including carcinoma-associated fibroblasts and pericytes, but not by cancer cells themselves in human colon cancer tissues. Blockade of PDGF-R signaling pathways in tumor-associated stromal cells has been shown to inhibit the stromal reaction. Activation of phosphoinositide 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling is frequently observed in many cancer types and has been implicated in the pathogenesis of various diseases. mTOR is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell proliferation, metabolism, survival, and angiogenesis. However, the effects of mTOR inhibitors on the stromal component of tumor tissues has not been studied. In the present study, the effects of PDGF-R tyrosine kinase inhibitors (nilotinib) and mTOR inhibitor (everolimus) on tumor development and spread, were examined using an orthotopic model of human colon cancer, whereby KM12SM cells were implanted into the cecum of nude mice. Groups of mice (n=7 each) received saline (control), nilotinib (100mg/kg/day) or everolimus (1mg/kg/day), or a combination of nilotinib (100mg/kg/day) and everolimus (1mg/kg/day) by daily oral gavage. After 4 weeks of treatment, tumors and lymph nodes were resected, and specimens were analyzed histologically and immunohistochemically for comparison between the treatment group and control group. After treatment with nilotinib(vs. saline), the stromal reaction was significantly decreased. After treatment with everolimus, the stromal reaction was not decreased, but tumor cell proliferation and microvessel density were decreased, and the proportion of apoptotic tumor cells was increased. With the combination therapy, both tumor cell proliferation and the stromal reaction were decreased and apoptosis of tumor cells was increased, resulting in a remarkable decrease in tumor size and the number of metastatic lymph nodes. Therefore, concurrent inhibition of tumor cells and activated stromal cells by combination therapy using PDGF-R tyrosine kinase inhibitor and mTOR inhibitor may represent a novel molecular-targeted approach for patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuhisa Chayama. Blockade mTOR and PDGF pathways by combination of molecular-targeted drugs inhibited cancer-stromal cell interaction in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2074. doi:10.1158/1538-7445.AM2013-2074

Abstract 4004: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Bone marrow-derived mesenchymal stem cells (MSCs) are reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. Furthermore, the migratory and invasive abilities of KM12SM colon cancer cells were shown by Transwell invasion assay to be enhanced by indirect co-culture with MSCs. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of metallothionein (MT) mRNA increased markedly by indirect co-culture of KM12SM cells with MSCs. Additionally, in an orthotopic nude mouse model of colon cancer, MT mRNA expression levels were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone. MTs comprise a family of low-molecular-weight, cysteine-rich intracellular proteins that are associated with protection against DNA damage, oxidative stress, and apoptosis. The potential role of MTs in carcinogenesis remains unknown. We examined MT expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 101 tumors were analyzed. MT immunoreactivity was detected in tumor cells and in atypical stromal cells. Atypical stromal cells with strong MT expression were found in invasive cancers, at the invasive edge, but not in mucosal cancers or adenomas. Levels of MT expression in tumor cells correlated inversely with disease stage. These findings suggest that the interaction between MSCs recruited into tumor stroma and the tumor cells of invasive colon cancers induces and depends on MT expression. Further studies are needed to clarify the specific effects of MTs on tumor cells and stromal cells. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Ryo Yuge, Mieko Onoyama, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2013-4004

Abstract 1509: Modulation of stromal reaction by PDGF-R tyrosine kinase inhibitor in an orthotopic mice model of gastric carcinoma

Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. Many human tumors secrete platelet-derived growth factor (PDGF) ligands, and PDGF receptors (PDGF-Rs) are expressed by various stromal cell populations, including carcinoma-associated fibroblasts (CAFs). We reported recently that, in a mice model of colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) traveled to tumor stroma, where they differentiated into CAF-like cells. In the present study, we examined PDGF-B and PDGF-Rβ expression in relation to clinicopathologic features of human gastric carcinomas. We also examined the effects of PDGF-R tyrosine kinase inhibitors (imatinib, nilotinib) on tumor stroma in an orthotopic nude mouse model of human gastric carcinoma. Five gastric carcinoma cell lines and 38 surgical specimens of gastric carcinoma were used. Under culture conditions, gastric cancer cell lines expressed PDGF-B at various levels but not PDGFR-β. When cells from these lines were implanted orthotopically into the gastric wall of nude mice, cells with high PDGF-B expression resulted in tumors with abundant stroma. In contrast, cells with low PDGF-B expression resulted in medullary tumors with little stromal reaction. In surgical specimens, we found PDGF-B expression to be associated with stromal reaction. PDGF-B and PDGF-Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens and orthotopic tumors, tumor cells expressed PDGF-B, but PDGF-Rβ was expressed predominantly by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells. Treatment with PDGF-R tyrosine kinase inhibitors did not inhibit tumor growth but did significantly decrease the stromal reaction, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In addition, tumor tropism of MSCs was also inhibited by treatment with PDGF-R inhibitors in vivo and in vitro. The PDGF/PDGF-R signaling pathway appears to regulate cancer-stromal cell interaction, and target molecule-based inhibition of the stromal reaction may hold promise as an effective anti-tumor therapy. 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 1509. doi:1538-7445.AM2012-1509