The homeobox gene MSX2 determines chemosensitivity of pancreatic cancer cells via the regulation of transporter gene ABCG2.

Pancreatic cancer is one of the life-threatening cancers due to the difficulty in the curative surgery and resistance against conventional therapeutic strategies. Recent studies indicated that cancer stem cells, which exist as a small number of cells within the entire cancer tissue, contribute to the disease progression. Cancer stem cells reveal resistance against conventional chemotherapy, which is derived from the high-expression of multiple transporter genes. Our previous study demonstrated the aggravating role of the homeobox gene MSX2 as an inducer of epithelial-mesenchymal transition, and MSX2 turned out to correlate with the chemoresistance in the current study. Comprehensive analysis of the MSX2-target gene has identified ABCG2 as the responsible gene. Since previous studies reported the pivotal role of ABCG2 as a determining factor of cancer stem cells, the detailed regulatory mechanism of ABCG2 expression by MSX2 was investigated. As a result, the MSX2 expression level in each cell line well correlated with the ABCG2 expression level, and alteration of the MSX2 expression level by over-expression or siRNA-based knockdown affected the ABCG2 expression accordingly. Finally, we identified the functional cooperation of MSX2 and SP1 in the transcriptional regulation of ABCG2 via the SP1 binding elements within the ABCG2 promoter. These findings clarified the intriguing regulatory mechanism of the cancer stem cell-related gene, and will delineate a novel therapeutic target in pancreatic cancer.

Calcium-binding protein S100P is a novel diagnostic marker of cholangiocarcinoma.

The incidence and mortality of cholangiocarcinoma are increasing despite improvements in the diagnostic method. Since the sensitivity of brushing cytology for cholangiocarcinoma is not satisfactory, a novel diagnostic marker needs to be established. A recent report has suggested upregulation of the calcium-binding protein S100P in cholangiocarcinoma. The expression status of S100P in normal bile duct and cholangiocarcinoma tissues was assessed by immunohistochemistry. The expression levels of S100P mRNA in the brushing cytology samples during endoscopic retrograde cholangiopancreatography (ERCP) from benign biliary strictures and cholangiocarcinoma were assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The sensitivity and specificity of each diagnostic strategy was compared. S100P was frequently expressed in the cholangiocarcinoma tissues, but not in the normal bile duct. The brushing cytology samples from the cholangiocarcinoma cases revealed higher expression levels of S100P compared with the benign biliary strictures. The relative expression level of S100P could determine the cholangiocarcinoma at higher sensitivity than classical cytology, and the combination of the S100P expression level and cytology yielded a sensitivity of 90.0%, with a specificity of 92.0%. Calcium-binding protein S100P is a novel marker of cholangiocarcinoma. Detecting the S100P expression levels in brushing cytology samples has a diagnostic value, which will be helpful for better diagnosis of cholangiocarcinoma.

Granular cell tumor of the pancreas: A case report and review of literature.

Granular cell tumors, also called Abrikossof's tumors, were originally described by Abrikossof A in 1926. The first case of a pancreatic granular cell tumor was described in 1975 and only 6 cases have been reported. We describe a case of granular cell tumor in the pancreas showing pancreatic duct obstruction. Because imaging studies showed findings compatible with those of pancreatic carcinoma, the patient underwent distal pancreatectomy. Histological examination showed that the tumor consisted of a nested growth of large tumor cells with ample granular cytoplasm and small round nuclei. The tumor cells expressed S-100 protein and were stained with neuron-specific enolase and periodic acid-Schiff, but were negative for desmin, vimentin, and cytokeratin. The resected tumor was diagnosed as a granular cell tumor. To our knowledge, this is the seventh case of Granular cell tumor of the pancreas to be reported.

Prediction of invasive carcinoma in branch type intraductal papillary mucinous neoplasms of the pancreas.

BACKGROUND: Patients with branch duct type intraductal papillary mucinous neoplasm (BD-IPMN) without invasion usually show favorable prognosis. However, the prognosis becomes poor when the IPMN lesions give rise to invasive carcinoma cells. In addition, recent studies have revealed that BD-IPMN is frequently complicated by common type pancreatic ductal carcinoma. Thus, the prognosis of BD-IPMN depends on the occurrence of these two types of invasive carcinoma. However, little is known about the risk factors for the development of these invasive carcinomas in BD-IPMN. This study aims to identify the factors which predict the development of invasive carcinoma in BD-IPMN. METHODS: Invasive pancreatic carcinoma associating with BD-IPMN was classified as invasive IPMN group (invasive carcinoma derived directly from IPMN lesions) and concomitant group (common type of invasive carcinoma concomitant with BD-IPMN). The relation between the incidence of each type of invasive carcinoma in BD-IPMN and the clinicopathological parameters was retrospectively analyzed. RESULTS: There were 12 patients with invasive IPMN and 7 patients with concomitant cancer in 159 patients with BD-IPMN. Diameter of dilated branch (P < 0.001) or main pancreatic duct (MPD) (P = 0.001), size of mural nodule (P < 0.001), serum CEA level (P < 0.001) and serum CA19-9 level (P < 0.001) were factors associated significantly with invasive IPMN by univariate analysis. Among these factors, mural nodule with size larger than 6.5 mm [odds ratio 14.86 (95% CI 1.37-60.45); P = 0.02] and serum carcinoembryonic antigen (CEA) level over 5 ng/ml [odds ratio 6.91 (95% CI 1.17-54.13); P = 0.03] were found to be the factors independently associated with invasive IPMN. On the other hand, both univariate and multivariate analyses revealed that elevated carbohydrate antigen 19-9 (CA 19-9) levels were associated with the occurrence of concomitant ductal carcinoma in BD-IPMN [odds ratio 10.31 (95% CI 1.77-81.51); P = 0.01]. CONCLUSIONS: Our results suggested that careful imaging study of the entire pancreas in addition to tumor lesions and measurement of serum CEA and CA19-9 would be required to find out the development of the two types of invasive carcinoma in BD-IPMN.

Expression of MSX2 predicts malignancy of branch duct intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: To distinguish malignant from benign branch duct (BD)-intraductal papillary mucinous neoplasm (IPMN) still remains difficult. Recently, we revealed that MSX2 was frequently expressed in pancreatic cancer and its expression was correlated with aggressive behavior of the cancer. The aim of this study was to assess the involvement of MSX2 in IPMN development and whether its expression would differentiate malignant from benign IPMN. METHODS: Seventeen microdissected lesions and 45 IPMN tissues were used for quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. The role of MSX2 in the pancreatic duct cell was assessed by the induced expression of MSX2 in a normal human pancreatic duct epithelial cell line (HPDE). RESULTS: Malignant IPMN expressed significantly higher levels of MSX2 mRNA than benign IPMN lesions. MSX2 protein expression was frequently found in borderline and malignant lesions (20/29, 68.9%), while its expression was seen in only one of 16 benign IPMN tissues. Univariate analysis showed that nodules of 6 mm or more and MSX2 expression were significantly correlated with the malignancy of BD-IPMN (P = 0.022 and 0.0026, respectively), and multivariate analysis revealed that only MSX2 expression was identified as an independent factor to predict malignant BD-IPMN. HPDE cells expressing MSX2 showed increased cellular proliferation compared to control cells. CONCLUSIONS: Based on our results, MSX2 plays a pivotal role in the development of IPMN through growth stimulation of tumor cells, and its expression was identified as an independent predictive factor for malignancy of BD-IPMN.

Expression of the calcium-binding protein S100P is regulated by bone morphogenetic protein in pancreatic duct epithelial cell lines.

We previously reported that bone morphogenetic protein (BMP)-4 induces epithelial-mesenchymal transition in a pancreatic cancer cell line. To further investigate the detailed molecular mechanism of BMP action in pancreatic cancer, we carried out comprehensive microarray analysis in Panc-1 cells. The microarray analysis elucidated novel BMP target genes, and among them, the calcium-binding protein S100P was identified as an upregulated gene. S100P induction by BMP4 was confirmed by real-time reverse transcription-polymerase chain reaction and western blot analysis in Panc-1 and HPDE cells. Short interfering RNA-based knockdown of S100P expression sufficiently repressed BMP4-induced cell migration in Panc-1 cells. Because Panc-1 and HPDE cells express wild-type Smad4, we hypothesized that Smad4 might be indispensable for S100P induction by BMP4. S100P induction by BMP4 was not observed in the Smad4-null cell line BxPC3, and was sufficiently attenuated in short interfering RNA-based Smad4-knockdown Panc-1 cells. Interestingly, detailed promoter analysis revealed that upregulation of S100P by BMP4 was independent of the Smad-binding element, indicating that an additional unknown downstream factor of the Smad4-dependent pathway is necessary for this induction. These findings are the first of their kind, and this Smad4-dependent regulation of S100P by BMP signaling might explain the migratory mechanism of cancer cells, which is still unknown.

Periostin, secreted from stromal cells, has biphasic effect on cell migration and correlates with the epithelial to mesenchymal transition of human pancreatic cancer cells.

Periostin is a secretory protein that has been suggested to function as a cell adhesion molecule and promote the invasiveness or growth rate of tumors. However, little is known about the association of its expression and epithelial to mesenchymal transition (EMT), which is considered to play a crucial role in cancer cell metastasis. Thus, the authors investigated whether periostin could be involved in the process of EMT and the role of this gene in pancreatic cancer development. The expression of periostin was observed mainly in stromal cells but very little in cancer cells by immunohistochemistry and real-time RT-PCR. In vitro, pancreatic stellate cells (PSCs) exhibited a much higher basal expression of periostin compared with cancer cells. Periostin secreted in the supernatant from 293T cells that expressed periostin (approximately 150 ng/ml) inhibited the migration of pancreatic cancer cells. Coculture assay revealed that periostin expression in PSC was induced by pancreatic cancer cells. To assess the direct role of periostin in pancreatic cancer cells, the authors generated pancreatic cancer cell lines that stably express periostin. The induced expression of periostin (to 150 ng/ml) altered the morphology of cancer cells, changing them from mesenchymal to epithelial phenotypes with the induction of epithelial markers and a reduction of mesenchymal markers, and showed reduced cell migration in vitro and formed smaller tumors as well as suppressed metastasis in vivo. On the other hand, high concentration of recombinant periostin (1 microg/ml) promoted cell migration with AKT activation. The findings suggest that periostin has biphasic effect on the development of pancreatic cancer.

Expression of Sonic hedgehog signaling pathway correlates with the tumorigenesis of intraductal papillary mucinous neoplasm of the pancreas.

Sonic hedgehog (SHH) is frequently expressed in pre-cancerous lesions and carcinoma of the pancreas. A recent study revealed that its expression was higher in the intraductal papillary mucinous neoplasm (IPMN) of the pancreas than in the pancreatic carcinoma. However, the correlation between its signaling pathway and tumorigenesis of IPMN has not yet been well documented. We investigated the expression of mRNA and protein of SHH as well as its downstream transcription factor Gli1 in 19 microdissected lesions from 15 cases and in 75 lesions from 33 cases of the IPMN by one-step quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. SHH and Gli1 mRNAs were detected in all the examined lesions and 8 out of 19 lesions in IPMNs, respectively. SHH and Gli1 mRNAs were likely to be up-regulated from the adenoma and from borderline to carcinoma cells, respectively. Immunohistochemical analysis also reported that SHH and Gli1 expression was correlated with the grade of cell atypia. These findings suggested that HH signaling was activated in IPMNs and contributed to tumorigenesis in these types of neoplasms.

Up-regulation of MSX2 enhances the malignant phenotype and is associated with twist 1 expression in human pancreatic cancer cells.

MSX2 is thought to be a regulator of organ development and a downstream target of the ras signaling pathway; however, little is known about the role of MSX2 in the development of pancreatic cancers, most of which harbor a K-ras gene mutation. Therefore, we examined whether the presence of MSX2 correlates with the malignant behavior of pancreatic cancer cells. BxPC3 pancreatic cancer cells that stably overexpress MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration, and anchorage-independent cell growth were enhanced in MSX2-expressing cells. Injection of MSX2-expressing cells into the pancreas of nude mice resulted in a significant increase in liver metastases and peritoneal disseminations compared with injection of control cells. Microarray analysis revealed a significant induction of Twist 1 expression in cells that express MSX2. When MSX2 was inactivated in pancreatic cancer cells following transfection with an MSX2-specific small interfering RNA, Twist 1 was down-regulated. Immunohistochemistry of human pancreatic carcinoma tissue revealed that MSX2 was frequently expressed in cancer cells, and that increased expression of MSX2 significantly correlated with higher tumor grade, vascular invasion, and Twist 1 expression. These data indicate that MSX2 plays a crucial role in pancreatic cancer development by inducing changes consistent with epithelial to mesenchymal transition through enhanced expression of Twist 1.

The expression of MUC4 and MUC5AC is related to the biologic malignancy of intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas show heterogeneous proliferations with latent malignancy. Mucins (MUC) are high-molecular-weight glycoproteins, with an aberrant expression profile in various malignancies. Recently, MUC4 and MUC5AC expressions have been demonstrated to correlate with the unfavorable and the favorable prognosis of pancreatic duct cell carcinoma, respectively. However, little is known about these mucin expressions in IPMNs. METHODS: To clarify the role of MUC4 and MUC5AC expressions in IPMNs, the expression profiles of MUC4 and MUC5AC were investigated in 50 lesions from 17 specimens with 16 IPMNs by immunohistochemistry, using each of their specific antibodies. RESULTS: The expression of MUC4 was found in the lesions ranging from adenoma to cancer lesions of IPMNs, whereas it was undetectable in normal and hyperplastic lesions. Frequent expression of MUC4 is found in the higher grade of IPMNs (borderline and cancer lesions; 16/18 lesions, 94%). The differences were independently significant (P < 0.001) when the cutoff point was set between adenoma and borderline IPMNs. Similarly, frequent expression of MUC5AC was detected in the lesions from adenoma to cancer of IPMNs (32/34, 94%), whereas no intense expression was detected in normal or hyperplastic lesions. The significant difference was found when the cutoff point was set between hyperplasia and adenoma of IPMNs (P < 0.001). CONCLUSIONS: These results indicated that the expressions of MUC4 and MUC5AC are potential markers to distinguish adenoma or above malignant lesions of IPMNs from lesser malignant ones, respectively.

Immunoglobulin G4-related lymphoplasmacytic sclerosing cholangitis that mimics infiltrating hilar cholangiocarcinoma: part of a spectrum of autoimmune pancreatitis?

BACKGROUND: Autoimmune pancreatitis has been designated as sclerosing pancreatocholangitis, because this disease shows a high prevalence of bile-duct lesions. We present herein the clinical characteristics of unusual cases that show dominant bile-duct lesions and mimicking infiltrating hilar cholangiocarcinomas. METHODS: Clinical and pathologic findings of 3 patients with immunoglobulin (Ig) G4 related sclerosing cholangitis who had no apparent pancreatic lesions comparable with autoimmune pancreatitis were analyzed. OBSERVATIONS: All patients were middle-aged or elderly individuals with slightly elevated serum IgG4 concentrations and showed long-segment narrowing of the bile-duct system, mimicking infiltrating hilar cholangiocarcinoma without significant pancreatic change. The first patient was treated with a corticosteroid, resulting in amelioration of the narrowing of the bile duct. The second patient underwent surgery based on a diagnosis of cholangiocarcinoma. In the third patient, the bile-duct stricture reversed spontaneously 1 month after the drainage procedure. Pathologic findings of the bile ducts for all patients disclosed significant lymphoplasmacytic infiltration, including abundant IgG4-bearing plasma cells. CONCLUSIONS: The use of IgG4 immunostaining in biopsy specimens of the bile duct may identify the presence of corticosteroid-responsive lymphoplasmacytic sclerosing cholangitis.

Hepatic hydrothorax in the absence of ascites diagnosed by intraperitoneal spraying of indocyanine green.

Hepatic hydrothorax in the absence of ascites is a rare complication of liver cirrhosis. A 56-year-old woman was referred to our hospital because of a massive pleural effusion on the right side, requiring continuous drainage. Although the patient was known to have chronic hepatitis C, she had no signs of hepatic failure including ascites. A laparoscopic examination revealed a nodular liver and a small volume of ascites in the peritoneal cavity. Indocyanine green sprayed into the intraperitoneal cavity was excreted from the pleural drain just after the spraying, indicating an intraperitoneal origin of the pleural fluid. Discontinuation of pleural drainage and an introduction of standard treatment for ascites due to liver cirrhosis (including restriction of salt intake and diuretic administration) resulted in a marked decrease of pleural effusion.