Hiroshi Sawayama

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.

Human Microbiome Fusobacterium Nucleatum in Esophageal Cancer Tissue Is Associated with Prognosis.

Purpose: Fusobacterium nucleatum (F. nucleatum) is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. Although there are several reports of the relationship between F. nucleatum and the clinical outcome in human cancers, its prognostic significance in esophageal cancer remains unclear. Experimental Design: We quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. Significant pathways in F. nucleatum–positive esophageal cancer tissues were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using microarray data. Results: Esophageal cancer tissues contained significantly more F. nucleatum DNA than matched normal esophageal mucosa (P = 0.021; n = 60). F. nucleatum DNA was detected in 74 of 325 cases (23%). F. nucleatum DNA positivity was significantly associated with tumor stage, but not with sex, age, performance status, tobacco use, alcohol use, histology, tumor location, or preoperative treatment. F. nucleatum DNA positivity was also significantly associated with cancer-specific survival [log-rank P = 0.0039; univariate HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.031]. The top-ranked KEGG pathway in F. nucleatum–positive tissues was “cytokine–cytokine receptor interaction.” A significant relationship between F. nucleatum and the chemokine CCL20 was validated by IHC. Conclusions: F. nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. F. nucleatum might also contribute to aggressive tumor behavior through activation of chemokines, such as CCL20. Clin Cancer Res; 22(22); 5574–81. ©2016 AACR.

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.

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.

The microbiome and hepatobiliary-pancreatic cancers.

The human intestinal microbiome encompasses at least 100 trillion microorganisms that can influence host immunity and disease conditions, including cancer. Hepatobiliary and pancreatic cancers have been associated with poor prognosis owing to their high level of tumor invasiveness, distant metastasis, and resistance to conventional treatment options, such as chemotherapy. Accumulating evidence from animal models suggests that specific microbes and microbial dysbiosis can potentiate hepatobiliary-pancreatic tumor development by damaging DNA, activating oncogenic signaling pathways, and producing tumor-promoting metabolites. Emerging evidence suggests that the gut microbiota may influence not only the efficacy of cancer chemotherapies and novel targeted immunotherapies such as anti-CTLA4 and anti-CD274 therapies but also the occurrence of postoperative complications after hepatobiliary and pancreatic surgery, which have been associated with tumor recurrence and worse patient survival in hepatobiliary-pancreatic cancers. Hence, a better understanding of roles of the gut microbiota in the development and progression of hepatobiliary-pancreatic tumors may open opportunities to develop new prevention and treatment strategies for patients with hepatobiliary-pancreatic cancer through manipulating the gut microbiota by diet, lifestyle, antibiotics, and pro- and prebiotics.

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.

The role of intestinal bacteria in the development and progression of gastrointestinal tract neoplasms

More than 100 trillion microorganisms inhabit the human intestinal tract and play important roles in health conditions and diseases, including cancer. Accumulating evidence demonstrates that specific bacteria and bacterial dysbiosis in the gastrointestinal tract can potentiate the development and progression of gastrointestinal tract neoplasms by damaging DNA, activating oncogenic signaling pathways, producing tumor-promoting metabolites such as secondary bile acids, and suppressing antitumor immunity. Other bacterial species have been shown to produce short-chain fatty acids such as butyrate, which can suppress inflammation and carcinogenesis in the gastrointestinal tract. Consistent with these lines of evidence, clinical studies using metagenomic analyses have shown associations of specific bacteria and bacterial dysbiosis with gastrointestinal tract cancers, including esophageal, gastric, and colorectal cancers. Emerging data demonstrate that intestinal bacteria can modulate the efficacy of cancer chemotherapies and novel targeted immunotherapies such as anti-CTLA4 and anti-CD274 therapies, the process of absorption, and the occurrence of complications after gastrointestinal surgery. A better understanding of the mechanisms by which the gut microbiota influence tumor development and progression in the intestine would provide opportunities to develop new prevention and treatment strategies for patients with gastrointestinal tract cancers by targeting the intestinal microflora.

Treatment results of FOLFOX chemotherapy before surgery for lymph node metastasis of advanced colorectal cancer with synchronous liver metastasis: the status of LN metastasis and vessel invasions at the primary site in patients who responded to FOLFOX

PurposeThe combination of chemotherapy and surgery holds promise for improving CRC patient prognosis. We evaluated the pathological impact of chemotherapy on primary lesions and lymph node (LN) metastases retrospectively.MethodsSixteen CRC patients with synchronous liver metastasis underwent a radical operation between March 2005 and August 2007. Eight of the 16 cases (surgery group) were operated on for the primary lesion without chemotherapy and another 8 cases (chemotherapy group) were operated on after chemotherapy with FOLFOX (median: 8 courses).ResultsFive of the 8 patients in the surgery group were found to have pathological LN metastasis (62.5%; N0 37.5%, N1 37.5%, N2 25%). However, only 2 of the 8 patients in the chemotherapy group were found to have LN metastasis (25%; N0 75%, N1 25%, N2 0%). The ratio of LN metastasis (number of metastatic LNs/resected LNs in total) was 11.1% in the surgery group, but it was 4.8% in the chemotherapy group. Necrotic areas were widely detected in the LN specimens of the chemotherapy group. The percentage of lymphatic (ly) and vascular (v) invasion in the primary lesions was smaller in the chemotherapy group (ly 12.5% vs. 25.0%) than in the surgery group (ly 62.5% vs. 50.0%). The patients in the chemotherapy group had no significant adverse effects and did not show an worse survival rate overall than the surgery group.ConclusionsA promising effect of chemotherapy on the status of LN metastasis and vessel invasions at the primary site was observed in the patients who responded to FOLFOX.