Naotake Funamizu

MicroRNA-301b promotes cell invasiveness through targeting TP63 in pancreatic carcinoma cells.

Recent studies have demonstrated that deregulated microRNA (miR) expression is implicated in the development of human cancers. In the aberrant miR expression, miR-301 is upregulated in cancers, such as pancreatic, colorectal and oral carcinoma. Based on this evidence, we investigated the contribution of miR-301 to pancreatic carcinoma and the novel target genes of miR-301 in pancreatic carcinoma. In this study, we analyzed the effects of enforced and inhibited expression of miR-301b expression in the Panc-1 and BxPC-3 cell lines. MiR-301b expression levels were associated with cell invasiveness in both cell lines. Additional experiments indicated that miR-301b influences invasiveness through CDH1. Moreover microRNA target search algorithms and experimental strategies suggested that miR-301b suppressed TP63 expression as a novel target of miR-301b. Remarkably, miR-301b was also found to be associated with NF-κB activity in both cell lines. In summary, overexpressed miR-301b may suppress TP63 expression and contributes to promote cell invasiveness and to enhance gemcitabine resistance in pancreatic carcinoma cells. Thus, miR-301b may have potential as a novel therapeutic target for cancer treatment due to its stimulatory effects on cell invasiveness.

A Novel MIF Signaling Pathway Drives the Malignant Character of Pancreatic Cancer by Targeting NR3C2

Pancreatic cancers with aberrant expression of macrophage migration inhibitory factor (MIF) are particularly aggressive. To identify key signaling pathways that drive disease aggressiveness in tumors with high MIF expression, we analyzed the expression of coding and noncoding genes in high and low MIF-expressing tumors in multiple cohorts of pancreatic ductal adenocarcinoma (PDAC) patients. The key genes and pathways identified were linked to patient survival and were mechanistically, functionally, and clinically characterized using cell lines, a genetically engineered mouse model, and PDAC patient cohorts. Here, we report evidence of a novel MIF-driven signaling pathway that inhibits the orphan nuclear receptor NR3C2, a previously undescribed tumor suppressor that impacts aggressiveness and survival in PDAC. Mechanistically, MIF upregulated miR-301b that targeted NR3C2 and suppressed its expression. PDAC tumors expressing high levels of MIF displayed elevated levels of miR-301b and reduced levels of NR3C2. In addition, reduced levels of NR3C2 expression correlated with poorer survival in multiple independent cohorts of PDAC patients. Functional analysis showed that NR3C2 inhibited epithelial-to-mesenchymal transition and enhanced sensitivity to the gemcitabine, a chemotherapeutic drug used in PDAC standard of care. Furthermore, genetic deletion of MIF disrupted a MIF-mir-301b-NR3C2 signaling axis, reducing metastasis and prolonging survival in a genetically engineered mouse model of PDAC. Taken together, our results offer a preclinical proof of principle for candidate therapies to target a newly described MIF-miR-301b-NR3C2 signaling axis for PDAC management. Cancer Res; 76(13); 3838-50. ©2016 AACR.

Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels.

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.

MicroRNA-203 induces apoptosis by upregulating Puma expression in colon and lung cancer cells.

The present study investigated the relationship between microRNA-203 (miR-203) and the p53 upregulated modulator of apoptosis (Puma) in colon (HCT116) and lung cancer (A549) cells. Colon and lung cancer cell lines were selected for this study since a relationship between p53/miR-203 and p53/Puma has been established in both cancers. In the present study, adriamycin and nutlin-3 were used to activate p53, which induced both miR-203 and Puma expression in HCT116 cells. In contrast, HCT 116 cells with downregulated p53 showed decreased miR-203 and Puma expression. Importantly, we found that overexpressed miR-203 in HCT116 cells resulted in significantly increased Puma expression (P<0.05). Based on these findings, we hypothesized that another limb of the p53/Puma axis depends on miR-203 expression. To further validate this relationship, we used lung cancer cells (A549) and found that activated p53 increased both miR-203 and Puma expression. In addition, we found that Puma expression remained elevated in cells with overexpressed miR-203 in the presence of p53 downregulation. Cumulatively, our data purport that p53 not only increased Puma expression directly, but that it may also do so through miR-203. Additionally, functional studies revealed that miR-203 overexpression induced apoptosis and inhibited cell invasiveness.

Breast hemangioma with difficulty in preoperative diagnosis: a case report.

We report a rare case of breast hemangioma found in a 70-year-old Japanese female. Before seeking medical attention, the patient noticed a hard mass in her right breast but denied associated symptoms. A mammography revealed a well-circumscribed, highly dense, lobular nodule located in the middle inter portion of the right breast. To verify this finding, we used ultrasonography which revealed an irregular, iso-echoic nodule measuring 10 mm in the same portion. Based on these findings, we suspected a malignancy and performed a core needle biopsy. Unexpectedly, a histological examination of the biopsy displayed normal vasculature, adipose, and mammary tissues. In order to make an accurate diagnosis, the mass was surgically excised under general anesthesia and sent to pathology. Pathological findings of the mass were positive for breast hemangioma, and the patient has had no recurrence of the disease for the past 24 months.

Stent graft placement and balloon dilation for pseudoaneurysm complicated by distal arterial stenosis of the hepatic artery after pancreaticoduodenectomy

Hemorrhage from ruptured pseudoaneurysm is a rapidly progressing and potentially fatal complication after pancreaticoduodenectomy (PD). Stent graft placement for hepatic artery pseudoaneurysm has recently been reported as a valid alternative to transcatheter arterial embolization (TAE). We report a case of pseudoaneurysm of the common hepatic artery (CHA) with distal arterial stenosis treated by stent graft placement for pseudoaneurysm and balloon dilation for arterial stenosis due to pancreatic fistula after PD. A 67-year-old man underwent PD for intraductal papillary mucinous neoplasm with concomitant early gastric cancer. After the operation, pancreatic fistula developed, for which conservative management by drainage was continued. On the postoperative day 30, melena started. Emergency abdominal angiography revealed a pseudoaneurysm in the CHA, as well as distal arterial stenosis extending from the proper hepatic artery (PHA) to bilateral hepatic arteries. The portal vein was also stenotic due to pancreatic fistula, for which TAE was not judged suitable because of the risk of liver failure. Therefore, stent graft placement and balloon dilation were chosen. Three pieces of coronary covered stent were placed in a coaxial overlapping manner followed by balloon dilation of the proper and left hepatic arteries. Balloon dilation of the right hepatic artery failed by technical reasons. Completion arteriography confirmed the patency from the CHA to the left hepatic artery as well as the exclusion of the pseudoaneurysm. A liver abscess that developed in the right hepatic lobe after intervention was successfully treated by percutaneous drainage, and the patient discharged on day 27 after stent graft placement. Non-embolic management with preservation of the liver arterial flow may be an option for complicated pseudoaneurysm after PD.

Intestinal Obstruction Caused by Persimmon Bezoar: A Case Report.

Owing to their rare occurrence, persimmon bezoars are often overlooked as a cause of small bowel obstruction. We herein report a small bowel obstruction in a 67-year-old Japanese female who regularly consumed persimmons in autumn. The patient presented to our hospital with typical complaints of abdominal distension with pain for 2 days. Based on the patients history of a cesarean section 34 years ago, we initially diagnosed her with small bowel obstruction resulting from adhesions and placed an ileus tube. At first, the patient rejected the operation in spite of our recommendation. After 10 days, because the ileus tube was unable to relieve the obstruction, finally surgery was scheduled. Upon releasing the obstruction by partial resection of the small bowel, we found an impacted bezoar without any evidence of adhesions. After stone analysis, we first realized her regular persimmon intake. This case serves as an important reminder to obtain dietary history in order to investigate all possible causes of small bowel obstruction when intestinal obstruction is suspected.

Abstract 922: Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 (nuclear receptor subfamily group c member 2) in human pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy. Aberrant macrophage migration inhibitory factor (MIF) expression is associated with disease aggressiveness and increased MIF in PDAC predicts poor patient survival. Here we show that MIF-induced disease aggressiveness and poor survival in PDAC involves the inhibition of the nuclear receptor subfamily group c member 2 (NR3C2). Tumors with aberrant high levels of MIF (MIF-high) showed an increased expression of mir-301b and a decrease in NR3C2 expression as compared to the tumors with a lower level of MIF (MIF-low) in PDAC cases and a negative correlation existed between MIF and NR3C2 expressions in these tumors. Furthermore, patients with an increase in mir-301b or decrease in NR3C2 expression had a significantly shorter survival than other patients. MIF overexpression and knockdown confirmed that MIF up-regulates miR-301b, which then binds to the 3′UTR of NR3C2 and markedly decreases its expression. Moreover, MIF-overexpressing orthotopic tumor xenografts showed an increase in mir-301b and a decrease in NR3C2 expressions. MIF-induced regulation of mir-301b and NR3C2 functionally involved the PI3Kinase/Akt pathway. Examination of the tumor suppressor mechanism of NR3C2 revealed that it inhibits proliferation, colony formation, migration and invasion, and enhances sensitivity to chemotherapeutic drug gemcitabine in pancreatic cancer cells. Furthermore, NR3C2 knock down enhanced epithelial-to-mesenchymal transition. These data identify a novel MIF-induced signaling pathway targeting NR3C2, leading to increased disease aggressiveness and poor outcome in PDAC. We identified a MIF-driven signaling pathway that inhibits a previously undescribed tumor suppressor of PDAC, NR3C2, involving miR-301b. The inhibition of NR3C2 enhanced disease aggressiveness and predicted to poor survival, as shown by our survival analysis. Therapies that target the MIF-miR-301b-NR3C2 axis may improve disease outcome in pancreatic cancer. Citation Format: Shouhui Yang, Peijun He, Jian Wang, Aaron Schetter, Wei Tang, Naotake Funamizu, Jochen Gaedcke, Michael Ghadimi, Matthias Gaida, Thomas Ried, Nader Hannah, H. Richard Alexander, S. Perwez Hussain. Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 (nuclear receptor subfamily group c member 2) in human pancreatic 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 922. doi:10.1158/1538-7445.AM2015-922

Cholangiocarcinoma cell line TK may be useful for the pharmacokinetic study of the chemotherapeutic agent gemcitabine.

Cholangiocarcinoma is a disease with a poor prognosis. A human cholangiocarcinoma cell line, TK, was previously established to enable further understanding of the disease. We conducted this investigation to determine whether or not the TK line is useful for pharmacokinetic study of the chemotherapeutic agent gemcitabine (GEM). Along with the BXPC3 human pancreatic adenocarcinoma cell line, the sensitivity to and effects on the TK cell line of GEM were compared. The influence of deoxycytidine kinase (dCK) transduction was also comparatively investigated. The effects of GEM in terms of drug sensitivity of the TK cell line, cell cycle and levels of transcripts of key enzymes were comparable to the BXPC3 cell line. Responses to the drug were similar in both cell lines. In contrast to pancreatic carcinoma, cell lines for research on cholangiocarcinoma have been limited. This study suggests the application of the TK cell line to the pharmacokinetic study of the chemosensitization of therapeutic drugs, such as GEM.

Abstract 4363: Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 in human pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MIF is a pleiotropic cytokine, which plays a role in inflammatory and immune responses and is implicated in tumorigenesis. Our previous study showed that MIF enhances tumor aggressiveness and predicts outcome in patients with pancreatic ductal adenocarcinoma (PDAC). Aberrant microRNA (miRNA) expression is a common feature in cancer and cytokine responsive miRNAs are recently described as critical effectors in tumor progression. We have investigated MIF-associated miRNAs and their interactive roles in the progression of human pancreatic cancer. Global analysis of miRNA expression revealed 38 differentially expressed miRNAs in MIF-high as compared with MIF-low cases of PDAC based on the median value. Kaplan-Meier survival analysis showed that a higher expression of miR-301b in tumors was associated with poorer survival (p=0.008, N=37), which was further validated in an independent cohort of PDAC patients (p=0.021, N=42). Additionally, MIF expression positively correlated with miR-301b and mediated its expression in several human pancreatic cancer cells. Mechanistic analysis revealed that miR-301b promotes pancreatic cancer cell migration and invasion. Furthermore, nuclear receptor subfamily group c member 2 (NR3C2) was identified as a functional target of miR-301b. NR3C2 inhibited tumor migration and invasion and a higher expression of NR3C2 predicted better survival in pancreatic cancer patients. Further in vivo studies showed a significant decrease in NR3C2 in MIF-overexpressing orthotopic xenograft as compared to control. Taken together, our results demonstrate that MIF and miR-301b interactively contribute to pancreatic cancer progression by targeting NR3C2. This novel MIF-miR-301b-NR3C2 axis may provide potential targets for improving disease outcome in pancreatic cancer. Citation Format: Shouhui Yang, Peijun He, Jian Wang, Aaron Schetter, Naotake Funamizu, Katsuhiko Yanaga, Jochen Gaedcke, B. Michael Ghadimi, Matthias M. Gaida, Thomas Ried, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 in human pancreatic 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 4363. doi:10.1158/1538-7445.AM2014-4363