Impact of loss-of-function mutations at the RNF43 locus on colorectal cancer development and 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 © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin ß1 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 ß1 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 ß1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin ß1 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.

Duration of Smoking Cessation and Postoperative Morbidity After Esophagectomy for Esophageal Cancer: How Long Should Patients Stop Smoking Before Surgery?

BACKGROUND: Smoking is one of the risk factors for postoperative morbidity. There were no studies on the correlation between the duration of smoking cessation and the incidence of morbidities after esophagectomy. METHODS: A total of 246 patients undergoing elective esophagectomy with 2- or 3-field lymphadenectomy for esophageal cancer between April 2005 and February 2015 were retrospectively analyzed. Patients were divided into five groups according to the duration of smoking cessation [no smoking cessation (including cessation for a few days), cessation for 7-30, 31-90, ≥91 days, never smoker]. RESULTS: Any morbidity of Clavien-Dindo classification (CDc) ≥II, pneumonia, any pulmonary morbidity, surgical site infection, cardiovascular morbidity, and severe morbidities of CDc ≥IIIb frequently occurred in patients with no smoking cessation. The incidence of pneumonia and severe morbidities decreased as the duration of smoking cessation became longer. Logistic regression analysis suggested that no smoking cessation was the independent risk factor for any pulmonary morbidity (HR 3.68, 95% CI 1.152-11.74; p = 0.028). Smoking cessation ≤30 days was also the independent risk factor for pneumonia (HR 2.89, 95% CI 1.141-7.301; p = 0.025). Smoking cessation ≤90 days was the independent risk factor for severe morbidities of CDc ≥IIIb (HR 2.82, 95% CI 1.072-7.427; p = 0.036). CONCLUSIONS: Preoperative smoking cessation more than 90 days is ideal to reduce morbidities after esophagectomy. When patients with insufficient smoking cessation undergo esophagectomy, careful perioperative management is required.

Reconstruction Using a Pedunculated Gastric Tube with Duodenal Transection After Esophagectomy and Pharyngolaryngectomy.

BACKGROUND: Gastric conduit ischemia is sometimes correlated with anastomosis-related morbidities after esophagectomy and pharyngolaryngectomy.1 (-) 5 A lack of connection between the right and left gastroepiploic vessels and intraoperative injury to these vessels could cause conduit ischemia. In addition, tensioned anastomosis due to a short gastric tube also could contribute to anastomotic leaks. This report introduces a reconstruction technique using a pedunculated gastric tube with duodenal transection for these cases. METHODS: Creation of a gastric tube in the greater curvature of the stomach is performed with linear staplers. Only the right gastroepiploic vessels are preserved. The gastric tube is finally fashioned with a width of approximately 4 cm. The peripheral right gastroepiploic vessels to the pylorus are sacrificed. After the bulbs are transected, a pedunculated gastric tube is moved, with confirmation whether it has sufficient length for anastomosis in the neck. After the anal side of the gastric tube is transected, Roux-en-Y gastrointestinal anastomosis is performed. Finally, esophagogastric or pharyngogastric anastomosis is performed. RESULTS: Between November 2011 and September 2014, 18 patients underwent the reported reconstruction technique due to short gastric tubes in 10 patients and a lack of connection between the right and left gastroepiploic vessels in 8 patients. Anastomotic leaks occurred in three patients (16.7 %), conduit necrosis in no patients, and strictures in no patients, respectively. Two patients had an anastomotic grade 2 leak, and one patient had an anastomotic grade 3 leak. CONCLUSION: The current reconstruction technique is a good alternative for patients at risk of conduit ischemia and patients with a short gastric tube after esophagectomy and pharyngolaryngectomy.

A clinicopathological analysis of primary mucosal malignant melanoma.

PURPOSES: Primary mucosal malignant melanoma (PMMM) is a rare and highly lethal neoplasm associated with a poor prognosis. CXC chemokine receptor 4 (CXCR4) is expressed on various tumor cells, including malignant melanoma. Recent data indicate that CXCL12 and CXCR4 play a critical role in the behavior of cancer cells and in the survival of cancer patients. However, there has been no study that has addressed the expression and function of CXCR4/CXCL12 signaling in PMMM. METHODS: Immunohistochemical staining for CXCL12 and Ki67 in biopsy tissues from 10 cases of PMMM was performed. We analyzed the correlations between the clinicopathological features and expression levels of CXCL12 and Ki67. RESULTS: Six cases showed a high level of CXCL12 expression, while four cases had a low level of expression. High expression of CXCL12 correlated with a poor prognosis, although statistical significance was not reached (p = 0.054). Ki67 was highly expressed in five cases, while the expression in the other five cases was low. There was no correlation between the Ki67 expression and prognosis. CONCLUSIONS: The findings of this study suggest that CXCL12 expression may play an important role in the biological behavior of PMMM and may be associated with a poor prognosis of PMMM patients.

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.

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.

High expression of glucose transporter 1 on primary lesions of esophageal squamous cell carcinoma is associated with hematogenous recurrence.

PURPOSE: Glucose transporter type 1 (Glut1) plays a crucial role in cancer-specific metabolism to adapt to the rapid growth and tumor microenvironment in diverse malignant tumors. This study examined the clinical, pathological, and prognostic features of Glut1 expression on primary lesions of esophageal squamous cell carcinoma. METHODS: Immunohistochemical staining of Glut1 and CD34 was performed using paraffin-embedded sections of tissues obtained from 145 resectable esophageal squamous cell carcinoma patients without preoperative treatment. Microvessel density was calculated from CD34 staining. RESULTS: Glut1 positivity was observed in 41 patients (28.2 %) and associated with depth of invasion [odds ratio (OR) 2.984; 95 % confidence interval (CI) 1.208-7.371; P = 0.018] and vascular invasion (OR 2.771; 95 % CI 1.118-6.871; P = 0.028) in multivariate analysis. Glut1 positivity was a significant disadvantage to both relapse-free survival [hazard ratio (HR) 2.021; 95 % CI 1.100-3.712; P = 0.023] and esophageal cancer-specific survival (HR 2.223; 95 % CI 1.121-4.411; P = 0.022) in univariate Cox hazard analysis, but was not independently associated with relapse-free survival or cancer-specific survival in multivariate analysis. The relationship between Glut1 expression and first relapse site was investigated. Glut1 positivity was not associated with lymph node recurrence (HR 1.009; 95 % CI 0.402-2.530; P = 0.985) but was significantly associated with hematogenous recurrence (HR 3.701; 95 % CI 1.655-8.273; P = 0.001) in univariate Cox hazard analysis. Microvessel density was calculated to evaluate angiogenesis, and it was observed that Glut1 positivity was significantly associated with high microvessel density (P < 0.001). CONCLUSIONS: Glut1 expression was associated with hematogenous recurrence. The findings provide evidence of the significance of Glut1 expression as a biomarker.

Evaluation of the necessity of primary tumor resection for synchronous metastatic colorectal cancer.

PURPOSE: We evaluated the need for primary tumor resection in patients with colorectal cancer (CRC) and synchronous unresectable metastases who underwent chemotherapy, and identified the associations between the primary tumor characteristics and risk of intestinal obstruction or perforation. METHODS: We retrospectively analyzed the survival and complication rates of patients with synchronous metastatic CRC treated between April 2005 and December 2011. RESULTS: Of 131 patients, 68 underwent primary tumor resection before chemotherapy, and 63 were treated without resection before chemotherapy. The overall survival (OS) did not significantly differ between the two groups (log-rank P = 0.53). In the resection group, 12 patients (17.6%) developed postoperative complications. In the non-resection group, 16 patients (25.4%) required surgical intervention owing to obstruction or perforation during their treatment. Surgical intervention did not affect the OS. A circumferential tumor was a risk factor for obstruction or perforation of the colorectum in non-resected patients (odds ratio = 11.163; P = 0.006). CONCLUSION: Resection of primary tumors before chemotherapy is unnecessary in selected patients with synchronous metastatic colorectal cancer. A circumferential tumor is a risk factor for obstruction or perforation during chemotherapy in cases without primary tumor resection.

Laparoscopic removal of an ingested fish bone that penetrated the stomach and was embedded in the pancreas: a case report.

BACKGROUND: The gastrointestinal tract can occasionally be perforated or penetrated by an ingested foreign body, such as a fish bone. However, there are very few reported cases in which an ingested fish bone penetrated the gastrointestinal tract and was embedded in the pancreas. CASE PRESENTATION: An 80-year-old male presented with epigastric pain. Computed tomography of the abdomen showed a linear, hyperdense, foreign body that penetrated through the posterior wall of the gastric antrum. There was no evidence of free air, abscess formation, migration of the foreign body into the pancreas, or pancreatitis. As the patient had a history of fish bone ingestion, we made a diagnosis of localized peritonitis caused by fish bone penetration of the posterior wall of the gastric antrum. We first attempted to remove the foreign body endoscopically, but failed because it was not detected. Hence, an emergency laparoscopic surgery was performed. A linear, hard, foreign body penetrated through the posterior wall of the gastric antrum and was embedded in the pancreas. The foreign body was safely removed laparoscopically and was identified as a 2.5-cm-long fish bone. Intraperitoneal lavage was performed, and a drain was placed in the lesser sac. The patient recovered without complications and was discharged on the 7th postoperative day. CONCLUSION: Laparoscopic surgery could be performed safely for the removal of an ingested fish bone embedded in the pancreas.

A fascia lata free flap in pelvic exenteration for Fournier gangrene due to advanced rectal cancer: a case report.

BACKGROUND: Fournier gangrene due to advanced rectal cancer is a rapidly progressive gangrene of the perineum and buttocks. Emergency surgical debridement of necrotic tissue is crucial, and secondary surgery to resect tumors is necessary for wound healing. However, pelvic exenteration damages the pelvic floor, increasing the likelihood of herniation of internal organs into the infectious wound. The management of pelvic exenteration for rectal cancer with Fournier gangrene has not yet been established. We herein describe the use of a fascia lata free flap in pelvic exenteration for rectal cancer with Fournier gangrene. CASE PRESENTATION: A 66-year-old male who had undergone colostomy for large bowel obstruction due to advanced rectal cancer and continued chemotherapy was referred to our hospital for Fournier gangrene resulting from chemotherapy. Emergency surgical debridement was performed, and the infectious wound around the rectal cancer was treated with intravenous antibiotic agents postoperatively. However, the tumor was exposed by the wound, and exudate persisted. Pelvic exenteration was performed due to tumor infiltration into the bladder and prostate. Tumor resection resulted in a defect in the pelvic floor. A fascia lata free flap (15 × 9 cm) obtained from the left thigh was fixed to the edge of the peritoneum and ileal conduit to close the defect in the pelvic floor and prevent small bowel herniation into the resected space. There was no intraabdominal inflammation or bowel obstruction postoperatively, and outpatient chemotherapy was continued. CONCLUSIONS: Surgical repair with a fascia lata free flap to close the defect in the pelvic floor led to a good clinical outcome for pelvic exenteration in a patient with Fournier gangrene due to advanced rectal cancer.

Chronic inflammation with Helicobacter pylori infection is implicated in CD44 overexpression through miR-328 suppression in the gastric mucosa.

BACKGROUND: Infection with Helicobacter pylori is the main risk factor for development of gastric cancer. CD44 overexpression, especially that of variant 9 (CD44v9), has also been implicated in the local inflammatory response and metaplasia-carcinoma sequence in human stomach. We recently identified miR-328 as one of the microRNAs targeting CD44 in gastric cancer. The aim of the current study was to determine the relationship between miR-328 and CD44v9 expression in H. pylori-infected gastric mucosa during the development of preneoplastic lesions. METHODS: Immunohistochemical staining of myeloperoxidase and CD44v9 was performed using paraffin-embedded tissue sections obtained from 54 patients who underwent gastric resection without preoperative treatment. The levels of miR-328 expression in the gastric mucosa were measured in the same patients using quantitative reverse transcription polymerase chain reaction. RESULTS: Both infiltration of myeloperoxidase-positive inflammatory cells and expression of proinflammatory cytokines closely correlated with H. pylori infection in the cancer-afflicted gastric mucosa. High CD44v9 expression levels, identified in the gastric mucosa in 61 % of samples (33/54), correlated significantly with H. pylori infection in the gastric mucosa. Notably, high CD44v9 expression was significantly associated with low miR-328 expression, whereas low CD44v9 expression was significantly associated with high miR-328 expression. CONCLUSIONS: We showed that miR-328 downregulation and de novo expression of CD44v9 occurred in H. pylori-infected gastric mucosa adjacent to gastric cancer compared with gastric mucosa not infected with H. pylori adjacent to gastric cancer. CD44v9-overexpressing cells are known to acquire reactive oxygen species resistance; thus, these cells may avoid cell death caused by various stress inducers, which may be linked to the origin of gastric cancer development.

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

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 CXCL(high)-expressing CAFs invaded more than when co-cultured with CXCL(low)-expressing CAFs; these invasive properties were impeded by CXCR4 antagonist AMD3100. We demonstrated that AEG cells co-cultured with CXCL12(high) CAFs were significantly more invasive than those co-cultured with CXCL12(low) 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.

Relationship between LINE-1 hypomethylation and Helicobacter pylori infection in gastric mucosae.

The DNA methylation alterations occurring in human cancers have two types: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Recently, to assess global DNA methylation, long interspersed nucleotide element 1 (LINE-1) retrotransposons, constituting a substantial portion of the human genome, attracts much attention. The aim of the current study was to clarify the significance of LINE-1 methylation level for epigenetic field defects and the relationships among LINE-1 methylation level in gastric mucosae, clinical and pathological features, including infection by Helicobacter pylori (H. pylori), a bacterium implicated in gastric cancer. By bisulfite-PCR pyrosequencing, we quantified the LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues from 87 gastric cancer patients, in gastric mucosae from 17 autopsied individuals without gastric cancers and in 20 gastric fresh frozen samples from non-gastric cancer patients. LINE-1 methylation in the noncancerous gastric mucosae of gastric cancer patients was significantly higher than in cancer tissues (P = 0.0006), but significantly lower than in the gastric mucosae of the autopsied individuals (P = 0.026), suggesting the formation of epigenetic field defect in noncancerous gastric mucosae. Moreover, LINE-1 hypomethylation of noncancerous gastric mucosae in gastric cancer patients significantly correlated with H. pylori infection (P = 0.037). We prospectively confirmed the similar result in 20 non-gastric cancer patients (P = 0.010). LINE-1 hypomethylation of gastric mucosae significantly correlated with H. pylori infection, supporting the potential of LINE-1 methylation level as a surrogate marker of epigenetic field defects for gastric cancer cancerization, particularly induced by H. pylori.

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.

Clinical impact of the Warburg effect in gastrointestinal cancer (review).

Cancer cells exhibit altered glucose metabolism, termed the Warburg effect, which is described by the increased uptake of glucose and the conversion of glucose to lactate in cancer cells under adequate oxygen tension. Recent genetic and metabolic analyses have provided insights into the molecular mechanisms of genes that are involved in the Warburg effect and tumorigenesis. The aim of this review was to discuss significant molecular insights into clinical impacts of the Warburg effect such as oncogenic alterations and overexpression of transcriptional factors (c-Myc and hypoxia-inducible factor), metabolite transporters (glucose transporters) and glycolytic enzymes (hexokinases 2, pyruvate kinase M2, pyruvate dehydrogenase kinase, isozyme 1, lactate dehydrogenase A). Overexpression of transcriptional factors, metabolite transporters and glycolytic enzymes was associated with poor prognosis and may be associated with chemoradiotherapy resistance in multiple gastrointestinal cancer cell types. Novel small molecules targeting these enzymes or transporters exert anti-proliferative effects. Glycolytic enzymes and metabolite transporters may be significant biomarkers for predicting cancer prognosis and may be therapeutic targets in 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.

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.