Hidetaka Sugihara

Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in cancer cells and promote redox adaptation

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.

Small molecule agonists of PPAR-γ exert therapeutic effects in esophageal cancer

The transcription factor PPAR-γ plays various roles in lipid metabolism, inflammation, cellular differentiation, and apoptosis. PPAR-γ agonists used to treat diabetes may have utility in cancer treatment. Efatutazone is a novel later generation PPAR-γ agonist that selectively activates PPAR-γ target genes and has antiproliferative effects in a range of malignancies. In this study, we investigated PPAR-γ status in esophageal squamous cell carcinoma (ESCC) and investigated the antiproliferative effects of efatutazone. PPAR-γ was expressed heterogeneously in ESCC, in which it exhibited an inverse relationship with Ki-67 expression. PPAR-γ expression was associated independently with good prognosis in ESCC. Efatutazone, but not the conventional PPAR-γ agonist troglitazone, inhibited ESCC cell proliferation in vitro and in vivo. Mechanistic investigations suggested that efatutazone acted by upregulating p21Cip1 protein in the nucleus through inactivation of the Akt pathway and dephosphorylation of p21Cip1 at Thr145 without affecting the transcriptional activity of p21Cip1. We also found that treatment with efatutazone led to phosphorylation of the EGF receptor and activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, the combination of efatutazone with the antiepithelial growth factor receptor antibody cetuximab synergized to negatively regulate the phosphoinositide 3-kinase-Akt and MAPK pathways. Together, our results suggest that efatutazone, alone or in combination with cetuximab, may offer therapeutic effects in ESCC.

Current perspectives toward the identification of key players in gastric cancer microRNA dysregulation

Acquired genetic and epigenetic alterations in normal cells give rise to transformed cells, which lead to tumor development. Elucidation of the precise mechanisms underlying primary and metastatic tumor formation is required. MicroRNAs (miRNAs) are small noncoding RNAs that play a major role in post‐transcriptional gene regulation during various biological processes. Accumulating evidence suggests that dysregulation of miRNAs is intimately involved in the carcinogenesis, progression and metastasis of many cancers, including gastric cancers (GCs), while the alteration of certain miRNAs provides biomarkers to detect early GCs. This review summarizes the most recent findings into the mechanisms of miRNA‐mediated regulation of GCs, which will support the development of diagnostic biomarkers and novel therapeutic strategies.

Noncoding RNA expression aberration is associated with cancer progression and is a potential biomarker in esophageal squamous cell carcinoma

Esophageal cancer is one of the most common cancers worldwide. Esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancer in Eastern Asian countries. Several types of noncoding RNAs (ncRNAs) function as key epigenetic regulators of gene expression and are implicated in various physiological processes. Unambiguous evidence indicates that dysregulation of ncRNAs is deeply implicated in carcinogenesis, cancer progression and metastases of various cancers, including ESCC. The current review summarizes recent findings on the ncRNA-mediated mechanisms underlying the characteristic behaviors of ESCC that will help support the development of biomarkers and the design of novel therapeutic strategies.

Interaction between gastric cancer stem cells and the tumor microenvironment.

Gastric cancer (GC) remains a leading cause of cancer-related deaths worldwide. Cancer stem cells (CSCs) are selectively capable of tumor initiation and are implicated in tumor relapse and metastasis, thus, governing the prognosis of GC patients. Stromal cells and extracellular matrix adjacent to cancer cells are known to form a supportive environment for cancer progression. CSC properties are also regulated by their microenvironment through cell signaling and related factors. This review presents the current findings regarding the influence of the tumor microenvironment on GC stem cells, which will support the development of novel therapeutic strategies for patients with GC.

Cancer-associated fibroblast-derived CXCL12 causes tumor progression in adenocarcinoma of the esophagogastric junction

Abstract Although cancer-associated fibroblasts (CAFs) mainly produce CXCL12 and stimulate CXCL12/CXCR4 signaling in cancer cells, the significance of this interaction in adenocarcinoma of the esophagogastric junction (AEG) was unclear. This study investigated the functional characteristics of CAF-derived CXCL12 in AEG. Immunohistochemical staining for CXCL12 was performed on sections from 123 AEG patients and analyzed against clinicopathological data. Newly isolated CAFs and normal fibroblasts were examined for phenotype. An invasion assay was performed with AEG cells co-cultured with CAFs isolated from AEG. CXCL12 expression was significantly associated with age, depth of invasion, lymphatic invasion, and lymph node metastases. High CXCL12 expression significantly correlated with poor prognosis. Isolated CAFs had abundant α-smooth muscle actin expression and showed various CXCL12 expression patterns. Notably, AEG cells co-cultured with CXCLhigh-expressing CAFs invaded more than when co-cultured with CXCLlow-expressing CAFs; these invasive properties were impeded by CXCR4 antagonist AMD3100. We demonstrated that AEG cells co-cultured with CXCL12high CAFs were significantly more invasive than those co-cultured with CXCL12low CAFs and that high CXCL12 expression correlates with poor prognosis in AEG patients. CXCL12 derived from CAFs in tumor microenvironment stimulates CXCL12/CXCR4 signaling in AEG cells and promotes their invasive ability, resulting in tumor progression.

Impact of loss-of-function mutations at the RNF43 locus on colorectal cancer development and progression: Impact of RNF43 mutations on colorectal cancer progression

RNF43 mutations are frequently detected in colorectal cancer cells and lead to a loss of function of the ubiquitin E3 ligase. Here, we investigated the clinical significance of RNF43 mutations in a large Japanese cohort and the role of RNF43 at various stages of colorectal cancer development and progression. Mutation analysis of the RNF43 gene locus with pyrosequencing technology detected RNF43 hotspot mutations in one (0.88%) of 113 colorectal polyp cases and in 30 (6.45%) of 465 colorectal cancer cases. Moreover, patients with colorectal cancer harbouring mutated RNF43 experienced a higher recurrence rate than those harbouring non‐mutated RNF43. In addition, the growth of RNF43 wild‐type colorectal cancer cell lines was significantly increased by RNF43 silencing. We generated Rnf43 knockout mice in a C57BL/6 N background by using the CRISPR–Cas9 system. Although intestinal organoids from Rnf43 knockout mice did not show continuous growth in the absence of R‐spondin, an azoxymethane/dextran sodium sulphate mouse model demonstrated that tumours were markedly larger in Rnf43 knockout mice than in wild‐type mice. These findings provide evidence that Wnt signalling activation by RNF43 mutations during the tumourigenic stage enhances tumour growth and promotes a high recurrence rate in colorectal cancer patients. Copyright

Abstract 423: CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer

Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric 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 423. doi:10.1158/1538-7445.AM2015-423