Kiyokazu Nakajima

Overexpression of miR-200c Induces Chemoresistance in Esophageal Cancers Mediated Through Activation of the Akt Signaling Pathway

Purpose: To determine the relationship between resistance to chemotherapy and microRNA (miRNA) expression in esophageal cancer, we focused on miRNAs known to be associated with maintenance of stem cell function. Experimental Design: Using 98 formalin-fixed, paraffin-embedded samples obtained from patients with esophageal cancer who had received preoperative chemotherapy followed by surgery, we measured expression levels of several miRNAs that are considered to be involved in the regulation of stem cell function (e.g., let-7a, let-7g, miR-21, miR-134, miR-145, miR-155, miR-200c, miR-203, and miR-296) by real-time reverse transcriptase PCR. Then, we examined the relationship between miRNA expression and prognosis or response to chemotherapy. To investigate the mechanism of miRNA-induced chemoresistance, in vitro assays were carried out using esophageal cancer cells. Results: Analyses of the 9 miRNAs expression showed that overexpression of miR-200c (P = 0.037), underexpression of miR-145 (P = 0.023), and overexpression of miR-21 (P = 0.048) correlated significantly with shortened overall duration of survival. In particular, miR-200c expression correlated significantly with response to chemotherapy (P = 0.009 for clinical response; P = 0.007 for pathologic response). In vitro assay showed significantly increased miR-200c expression in cisplatin-resistant cells compared with their parent cells (∼1.7-fold). In anti-miR-200c–transfected cells, chemosensitivity to cisplatin and apoptosis after exposure to cisplatin was found to increase as compared with the negative control. Western blotting showed that knockdown of miR-200c expression was associated with increased expression of PPP2R1B, a subunit of protein phosphatase 2A, which resulted in reduced expression of phospho-Akt. Conclusions: Results of this study emphasized the involvement of miR-200c in resistance to chemotherapy among esophageal cancers and that this effect was mediated through the Akt pathway. Clin Cancer Res; 17(9); 3029–38. ©2011 AACR.

Let-7 Expression Is a Significant Determinant of Response to Chemotherapy through the Regulation of IL-6/STAT3 Pathway in Esophageal Squamous Cell Carcinoma

Purpose: Cisplatin-based chemotherapy is widely used for esophageal cancer, sometimes in combination with surgery/radiotherapy, but poor response to chemotherapy is not uncommon. The aim of this study was to examine whether miRNA expression is useful to predict the response to chemotherapy in patients with esophageal cancer. Experimental Design: Using pretreatment biopsy samples from 98 patients with esophageal cancer who received preoperative chemotherapy, we measured the expression level of several miRNAs whose expression was altered in cisplatin-resistant esophageal cancer cell lines compared with those parent cell lines and examined the relationship between the miRNA expression and response to chemotherapy. In vitro assays were conducted to clarify the mechanism of miRNA-induced changes in chemosensitivity. Results: The expression levels of 15 miRNAs were altered in cisplatin-resistant cells. Of these, low expression of let-7b and let-7c in before-treatment biopsies from 74 patients of the training set correlated significantly with poor response to chemotherapy, both clinically and histopathologically. Low expression of let-7c also correlated with poor prognosis (P = 0.032). The relationship between let-7b and let-7c expression and response to chemotherapy was confirmed in the other 24 patients of the validation set. In in vitro assay, transfection of let-7c restored sensitivity to cisplatin and increased rate of apoptosis after exposure to cisplatin. Let-7c directly repressed cisplatin-activated interleukin (IL)-6/STAT3 prosurvival pathway. Conclusions: Let-7 expression in esophageal cancer can be potentially used to predict the response to cisplatin-based chemotherapy. Let-7 modulates the chemosensitivity to cisplatin through the regulation of IL-6/STAT3 pathway in esophageal cancer. Clin Cancer Res; 18(18); 5144–53. ©2012 AACR.

Effects of Ghrelin Administration After Total Gastrectomy: A Prospective, Randomized, Placebo-Controlled Phase II Study

BACKGROUND & AIMS Body weight (BW) loss and reduction of blood ghrelin level are commonly observed after total gastrectomy (TG). A prospective study was designed to elucidate whether exogenous ghrelin administration prevents postoperative BW loss by improving appetite and oral food intake in patients with gastric cancer after undergoing TG. METHODS In this randomized phase II study, 21 patients undergoing TG were assigned to a ghrelin (11 patients) or placebo group (10 patients). They received intravenous infusion of synthetic human ghrelin (3 microg/kg) or saline twice daily for 10 days after starting oral food intake following surgery. Changes in BW, appetite visual analog scale score, food intake calories, body composition, basal metabolic rate, and various blood test results were evaluated. RESULTS Excluding one patient who developed profound diaphoresis during ghrelin infusion, 20 patients completed the study. Food intake and appetite were significantly higher with ghrelin compared with placebo (average, 13.8 vs 10.4 kcal/kg/day [P = .030] and 5.7 vs 3.9 cm [P = .032], respectively). BW loss was significantly lower in the ghrelin than in the placebo group (-1.4% vs -3.7%; P = .044). Fat mass, lean body mass, and basal metabolic rate decreased significantly in the placebo group; however, the reductions in lean body mass and basal metabolic rate were not significant in the ghrelin group, although that of fat mass was significant. CONCLUSIONS Short-term administration of synthetic ghrelin was safe and successfully lessened postoperative BW loss and improved appetite and food intake after TG.

A Novel Sphingosine 1-Phosphate Receptor Agonist, 2-Amino-2-propanediol Hydrochloride (KRP-203), Regulates Chronic Colitis in Interleukin-10 Gene-Deficient Mice

Current treatments for patients with Crohns disease (CD) are based on recent advances in elucidating the pathophysiology of the disease. A satisfactory therapeutic strategy has not been well established. A new sphingosine 1-phosphate (S1P) receptor agonist, 2-amino-2-propanediol hydrochloride (KRP-203), has been developed for immunomodulation in autoimmune diseases and organ transplantation. We aimed to evaluate the efficacy and potency of KRP-203 on the treatment of chronic colitis in an interleukin (IL)-10 gene-deficient (IL-10–/–) mouse model. KRP-203 agonistic activity on S1P receptor was assessed in vitro. KRP-203 was administered for 1 or 4 weeks to IL-10–/– mice with clinical signs of colitis. The histological appearance of the colon and the numbers, phenotype, and cytokine production of lymphocytes were compared with a control group. KRP-203 treatment was effective in preventing body weight loss in the IL-10–/– colitis model. One-week administration resulted in the sequestration of circulating lymphocytes within the secondary lymphoid tissues. After 4 weeks of treatment, highly significant reductions were observed in number of CD4+ T cell and B220+ B cell subpopulations in the lamina propria of the colon and peripheral blood. KRP-203 obviously inhibited the production of interferon-γ, IL-12, and tumor necrosis factor-α by the colonic lymphocytes, but had no influence on IL-4 production. KRP-203 significantly inhibits ongoing IL-10–/– colitis in part through decreasing the infiltration of lymphocytes at inflammatory sites and by blocking T-helper 1 cytokine production in the colonic mucosa. Therefore, the possibility arises that KRP-203 plays a potential role in control of chronic colitis.

Secondary mutations in the kinase domain of the KIT gene are predominant in imatinib-resistant gastrointestinal stromal tumor.

Although imatinib showed high activity for advanced gastrointestinal stromal tumor (GIST) and improved the prognosis of GIST patients, resistance to the drug appears with prolonged use. Mechanisms of acquired resistance are still under investigation. In the present study, we carried out histologic and genetic analysis of 45 secondary resistant lesions obtained from 25 Japanese GIST patients treated with imatinib. All resistant lesions showed viable tumor cells expressing KIT protein, whereas imatinib‐sensitive lesions did not. All pre‐imatinib samples have KIT mutations either in exon 9 (n = 3) or exon 11 (n = 22), identified in the KIT gene of corresponding resistant tumors. In addition to primary mutations, 33 out of 45 tumors (73%) showed secondary KIT mutations in the kinase domain of the KIT gene. Secondary mutations are missense mutations and are mostly located in the kinase domains of the same allele to the primary mutations (cis‐position). Resistant lesions showed monoclonal development of tumor cells. Taken together, additional cis‐positioned mutations in the kinase domains are a major cause of secondary resistance to imatinib in Japanese GIST patients. (Cancer Sci 2008; 99: 799–804)

Effects of ghrelin administration during chemotherapy with advanced esophageal cancer patients: a prospective, randomized, placebo-controlled phase 2 study.

Cisplatin reduces plasma ghrelin levels through the 5‐hydroxytryptamine (5‐HT) receptor. This may cause cisplatin‐induced gastrointestinal disorders and hinders the continuation of chemotherapy. The authors of this report conducted a prospective, randomized phase 2 trial to evaluate the effects of exogenous ghrelin during cisplatin‐based chemotherapy.

Role of multidrug resistance protein 2 (MRP2) in chemoresistance and clinical outcome in oesophageal squamous cell carcinoma.

Background:Although multidrug resistance protein 2 (MRP2) confers chemoresistance in some cancer types, its implication on oesophageal squamous cell carcinoma (ESCC) remains unclear.Methods:We evaluated MRP2 expression by immunohistochemistry and RT–PCR using 81 resected specimens from ESCC patients who did or did not receive neo-adjuvant chemotherapy (NACT), including 5-fluorouracil, doxorubicin, and cisplatin (CDDP). Correlation between MRP2 expression and response to chemotherapy was also examined in 42 pre-therapeutic biopsy samples and eight ESCC cell lines.Results:MRP2-positive immunostaining was more frequently observed in ESCCs with NACT than in those without NACT (27.3 vs 5.4%). The MRP2-positive patients showed poorer prognosis than MRP2-negative patients (5-year survival rate, 25.6 vs 55.7%). Concordantly, ESCC with NACT showed 2.1-fold higher mRNA expression of MRP2 than those without NACT (P=0.0350). In pre-therapeutic biopsy samples of patients with NACT, non-responders showed 2.9-fold higher mRNA expression of MRP2 than responders (P=0.0035). Among the panel of ESCC cell lines, TE14 showed the highest MRP2 mRNA expression along with the strongest resistance to CDDP. Inhibition of MRP2 expression by small-interfering RNA reduced chemoresistance to CDDP.Conclusion:Our data suggested that MRP2 is one of molecules, which regulate the sensitivity to chemotherapy including CDDP in advanced ESCC patients.

A simple and safe technique for esophagojejunostomy using the hemidouble stapling technique in laparoscopy-assisted total gastrectomy

In laparoscopy-assisted total gastrectomy, esophagojejunostomy is technically difficult. We describe a safe and simple technique for circular-stapled esophagojejunostomy. After mobilization of the stomach and the esophagus, a semicircumferential esophagotomy is made at the anterior esophageal wall. An anvil of a circular stapling device, secured with a Prolene suture (Ethicon, Inc, Somerville, NJ), is introduced via the esophagotomy. The suture is advanced anteriorly so that the center rod penetrates the esophageal wall. The esophagus is staple transected at this point. The circular-stapled esophagojejunostomy is then performed using the hemidouble stapling technique. Laparoscopy-assisted total gastrectomies were performed in 10 patients with gastric cancers. All patients were completed laparoscopically without any complications. The time of anvil placement was 9 minutes in median. Although a wound infection occurred in 1 patient, there were no major complications. There was no mortality in this series. Esophagojejunostomy using this technique is safe and simple. Its practical value is the elimination of the need for pursestring suture placement.

Randomized phase II study of clinical effects of ghrelin after esophagectomy with gastric tube reconstruction

BACKGROUND Ghrelin is a peptide hormone with pleiotropic functions including stimulation of growth hormone secretion and appetite, and its levels decrease after esophagectomy. The aim of this study was to evaluate whether exogenous ghrelin administration can meliorate the postoperative decrease of oral food intake and body weight, which are serious complications after esophagectomy. METHODS This prospective randomized, placebo-controlled, clinical trial assigned a total of 20 patients with thoracic esophageal cancer who underwent radical operation into either a ghrelin (n =10) or placebo (n =10) group. Synthetic human ghrelin (3 microg/kg) or 0.9% saline placebo was administered intravenously twice daily for 10 days from the day after the start of food intake. The primary end point was calories of food intake. Comparison of appetite and changes in weight and body composition were also made between the 2 groups. RESULTS Intake of food calories was greater in ghrelin group than placebo group (mean 874 vs 605 kcal per day; P =.015). The appetite score tended to be greater in ghrelin group than placebo group (P =.094). Loss of weight was less in ghrelin group (-1% vs -3%; P =.019) and this attenuation was due largely to a decrease of lean body weight loss (0% vs -4%; P =.012). No side effects were observed in either groups. CONCLUSION These preliminary results suggest that administration of ghrelin after esophagectomy increased oral food intake and attenuated weight loss together with maintenance of lean body weight.

High expression of Lin28 is associated with tumour aggressiveness and poor prognosis of patients in oesophagus cancer.

Background:Lin28 is a negative regulator of the tumour suppressor microRNA, let-7, suggesting its role in tumourigenesis. However, the clinical significance of Lin28 expression in oesophageal cancer has not been elucidated.Methods:Lin28 and Lin28B expression was examined by immunohistochemistry in 161 tissues from patients with oesophageal cancer who had undergone curative surgery. The relationship between the expressions of Lin28 and Lin28B and various clinicopathological factors was examined. In vitro assays were conducted to determine the role of Lin28 in aggressiveness of oesophageal cancers using oesophageal cancer cell line.Results:Lin28 and Lin28B were overexpressed in oesophageal cancer cells compared with non-cancerous epithelial cells, especially in the invasive front. High expression of Lin28 and Lin28B correlated significantly with lymph node metastasis and poor prognosis. High expression of Lin28B expression correlated significantly with low expression of let-7. Multivariate analysis also identified Lin28B expression as an independent prognostic factor. In vitro assays showed that the proliferative and invasive activities were significantly reduced in Lin28B-knockdown cells, compared with control cells.Conclusion:High expression of Lin28 is associated with poor prognosis and high tumour aggressiveness in oesophageal cancer and these effects are mediated through increased proliferation and invasiveness of oesophageal cancer cells.