Concurrent Overexpression of Two Hyaluronidases, KIAA1199 and TMEM2, Strongly Predicts Shorter Survival After Resection in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by accelerated hyaluronan metabolism. Our previous studies have shown increased expression of 2 newly identified hyaluronidases, KIAA1199 and transmembrane protein 2 (TMEM2), in PDAC. However, the relationship between these 2 hyaluronidases is unknown. In the present study, we investigated the correlation between KIAA1199 and TMEM2 expression in PDAC. METHODS: Using quantitative real-time reverse transcription polymerase chain reaction, we analyzed KIAA1199 and TMEM2 mRNA expression in 11 PDAC cell lines and frozen tissues from 12 patients with PDAC. We used immunohistochemistry to investigate expression patterns of KIAA1199 and TMEM2 in archival tissues obtained from 92 patients with PDAC who underwent surgical resection. We compared survival between 4 groups according to expression patterns of KIAA1199 and TMEM2. RESULTS: We found a significantly positive correlation between KIAA1199 and TMEM2 mRNA in PDAC cell lines and tissues. Immunohistochemical analysis found that median overall survival was 30.2 months in patients with low expression of KIAA1199 and TMEM2 and 12.5 months in those with high expression of both. Patients with high expression of KIAA1199 and TMEM2 had significantly shorter survival than other patient groups. CONCLUSIONS: Concurrent overexpression of these 2 hyaluronidases could be a strong prognostic marker in PDAC.

Hypoxia increases KIAA1199/CEMIP expression and enhances cell migration in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplasia and hypoxic microenvironment. Our previous reports demonstrated that hyaluronan (HA), especially low-molecular-weight HA, provides a favourable microenvironment for PDAC progression. However, the effect of hypoxia on HA metabolism remains unknown. Using quantitative real-time RT-PCR and western blot analysis, we analysed the changes in the expression of HA-synthesizing enzymes (HAS2 and HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic conditions. Hypoxia increased the mRNA and protein expression of KIAA1199, whereas it decreased HYAL1 expression. The expression of HAS3 was increased and HAS2 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined using a transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced the migratory ability of PDAC cells, which was inhibited by KIAA1199 knockdown. We also used immunohistochemistry to analyse the protein expression of hypoxia inducible factor (HIF) 1α and KIAA1199 in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

Overexpression of transmembrane protein 2 (TMEM2), a novel hyaluronidase, predicts poor prognosis in pancreatic ductal adenocarcinoma.

BACKGROUND: Abnormal metabolism of hyaluronan (HA), a major component of extracellular matrix, is a hallmark of cancer. Our previous studies have shown the importance of enzymes responsible for HA degradation in the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). In the present study, we investigated the expression and function of transmembrane protein 2 (TMEM2), a recently identified HA-degrading enzyme, in PDAC. MATERIALS & METHODS: We used immunohistochemistry to investigate expression patterns of TMEM2 in archival tissues obtained from 100 patients with PDAC who underwent surgical resection from 1982 to 2012. The correlations between TMEM2 expression and clinicopathological variables, including survival, were determined using univariate and multivariate analyses. The effect of TMEM2 on proliferation and migratory ability (measured using transwell cell migration assay) of PDAC cells was determined by TMEM2 knockdown with small-interfering RNA (siRNA). RESULTS: Immunohistochemical analysis revealed high expression of TMEM2 in 22 (22%) of 100 patients. The overall survival was significantly shorter in patients with high TMEM2 expression than in those with low expression (P = 0.013). Multivariate analysis identified high TMEM2 expression as an independent factor predicting poor prognosis (P = 0.011). Unexpectedly, knockdown of TMEM2 resulted in increased migratory ability of PDAC cells, which was associated with increased expression of KIAA1199, a potent HA-degrading enzyme shown to enhance cell migration. CONCLUSION: TMEM2 overexpression is associated with poor prognosis in PDAC patients. Targeted disruption of this molecule, however, could enhance the aggressiveness of PDAC cells through a possible interaction with KIAA1199.

4-Methylumbelliferone inhibits enhanced hyaluronan synthesis and cell migration in pancreatic cancer cells in response to tumor-stromal interactions.

Hyaluronic acid (HA) in tumor stroma promotes tumor invasion and progression. 4-Methylumbelliferone (4-MU) is a potent HA synthesis inhibitor. In the present study, the effects of 4-MU on enhanced HA synthesis and cell migration in pancreatic ductal adenocarcinoma (PDAC) cells, in response to co-culture with stromal fibroblasts, was investigated. The HA concentration was determined using ELISA and a Transwell migration assay was used to analyze cell migratory capability. The mRNA expression levels of hyaluronan synthases (HAS1, HAS2 and HAS3) were determined using the quantitative polymerase chain reaction. Co-culture between Panc-1 cells and stromal fibroblasts markedly increased cell migration in association with increasing HA production, which was markedly associated with an increase in HAS3 mRNA expression. Treatment with 4-MU markedly decreased the HA production and cell migration of Panc-1 cells in the co-culture system. The results of the present study suggested that interactions between PDAC cells and stromal fibroblasts increased HA production, resulting in a marked increase in migration of PDAC cells, and 4-MU may be used as a chemotherapeutic agent to inhibit the enhanced migration of PDAC cells in response to tumor-stromal interactions.

A Case of Retroperitoneal Paraganglioma.

We report a surgical case of retroperitoneal paraganglioma. A paraganglioma is a catecholamine-producing tumor originating in the chromaffin cells of the sympathetic ganglion. It is a kind of pheochromocytoma which occurs on the outside of the adrenal gland. The patient was a 72 year old male with a history of hypertension and a pacemaker implantation. A mass in the ventral side of the right iliopsoas muscle was detected during a routine contrasting computed tomography (CT) examination for checking his pacemaker. The mass was considered to be malignant, and a laparotomy and mass enucleation was performed. It was diagnosed as phaeochromocytoma, based on the pathology and immunestology of the excised specimen. The hypertension was cured soon after the surgery. Nine months after surgery, there is no evidence of any abnormality or recurrence. There is a previous report of a recurrence 25 years after surgery, so a careful follow-up of this patient will be necessary in the future.

KIAA1199 is induced by inflammation and enhances malignant phenotype in pancreatic cancer.

BACKGROUND: Recent evidence suggests a critical role of hyaluronan (HA), especially low-molecular-weight HA (LMW-HA), in the aggressive tumor phenotype. Increased expression of KIAA1199, a newly identified protein involved in HA degradation, has been reported in various cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about the functional significance of KIAA1199 in PDAC. METHODS: Using siRNA knockdown and forced expression models, we investigated the effects of KIAA1199 expression on malignant behaviors (proliferation, migration, and invasion) of PDAC cells. We also examined the effect of inflammation on the transcriptional regulation of KIAA1199 using a pro-inflammatory cytokine and anti-inflammatory agent. RESULTS: Knockdown of KIAA1199 expression using siRNA resulted in decreased cell migration and proliferation. On the other hand, forced expression of KIAA1199 using gene transduction significantly enhanced the migration and invasion. Importantly, increased KIAA1199 expression was associated with an increased level of LMW-HA in the conditioned medium. Exposure to a pro-inflammatory cytokine, interleukin-1ß, increased the KIAA1199 transcription and enhanced the migration. In contrast, treatment with NS-398, a cyclooxygenase-2 inhibitor, decreased the KIAA1199 expression and inhibited the migration. CONCLUSIONS: These findings suggest that increased KIAA1199 expression may contribute to the aggressive phenotype partly through increasing the LMW-HA concentration. Our present results also suggest a possible link between inflammation, induced KIAA1199 expression, and enhanced migration during PDAC progression.

KIAA1199/CEMIP/HYBID overexpression predicts poor prognosis in pancreatic ductal adenocarcinoma.

BACKGROUND: KIAA1199 (also known as CEMIP or HYBID), a newly identified protein involved in hyaluronan degradation, has been suggested to play a critical role in cancer progression. The aim of this study was to investigate the expression and functional significance of KIAA1199 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Using quantitative real-time RT-PCR, we analyzed KIAA1199 mRNA expression in 6 PDAC cell lines and frozen tissues from 14 patients with PDAC. We also used immunohistochemistry to analyze KIAA1199 protein expression in formalin-fixed, paraffin-embedded tissues from 98 patients with PDAC. The KIAA1199 expression pattern was then correlated with clinicopathological variables and patient outcome. The effect of KIAA1199 on migratory ability of PDAC cells was determined by KIAA1199 knockdown with small-interfering RNA (siRNA). RESULTS: The KIAA1199 mRNA expression was significantly higher in PDAC tissues than in the corresponding non-tumor tissues (P < 0.0001). Immunohistochemical analysis revealed high expression of KIAA1199 in 26 (26.5%) of 98 PDAC tissues. The overall survival was significantly shorter in patients with high KIAA1199 expression than in patients with low KIAA1199 expression (P = 0.0001). In multivariate analysis, high KIAA1199 expression (P = 0.003) and UICC stage (P = 0.003) were independent factors predicting poor prognosis. Furthermore, the KIAA1199 mRNA expression was higher in most PDAC cell lines and siRNA knockdown of KIAA1199 resulted in decreased migration. CONCLUSION: These findings suggest that overexpression of KIAA1199 may contribute to increased migration of PDAC cells and predict shorter survival after surgical resection.

Increased Expression of HYAL1 in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Increased production and processing (degradation) of hyaluronan (HA) is critical for cancer invasion and metastasis. Although HA is known to be overexpressed in pancreatic ductal adenocarcinoma (PDAC), little is known about the expression and biological significance of HA-degrading enzymes, hyaluronidases (HYALs), in PDAC. METHODS: Expression of HYALs mRNA was examined in PDAC cells by quantitative real-time RT-PCR. HYAL1 protein expression was examined in primary PDAC tumors by enzyme-linked immuno-sorbent assay. The migratory ability of PDAC cells was determined by a transwell cell migration assay. RESULTS: Screening of mRNA expression of three major HYAL genes (HYAL1, 2, and 3) identified HYAL1 as a gene overexpressed in PDAC cells. Treatment of PDAC cells with 5-aza-2'-deoxycytidine and/or trichostatin A further increased the HYAL1 expression, suggesting a possible involvement of epigenetic mechanisms in the transcriptional regulation of this gene. HYAL1 protein concentrations were significantly higher in primary PDAC tissues as compared with nontumor pancreatic tissues (P = 0.049). Importantly, inhibition of HYAL activity by dextran sulfate significantly inhibited the migration of PDAC cells showing strong HYAL1 expression (P = 0.002). CONCLUSIONS: These findings suggest that overexpression of HYAL1 is a common mechanism that may contribute to the aggressive phenotype of PDAC.

Preoperative factors predicting the need for additional ports during single-incision laparoscopic cholecystectomy.

INTRODUCTION: Single-incision laparoscopic cholecystectomy (SILC) is gaining popularity as a minimally invasive technique that provides some benefits in terms of improved cosmesis. However, the insertion of an additional port is required in a subset of cases. METHODS: We retrospectively analyzed 308 SILC procedures in patients with benign gallbladder diseases, except acute cholecystitis, to identify preoperative factors predicting the need for an additional port. RESULTS: SILC was completed with a single incision in 272 patients (88%); the insertion of at least one additional port was required in 36 patients (12%). The additional port requirement was associated with a longer operative time (P < 0.0001), greater intraoperative blood loss (P < 0.0001), and longer postoperative hospital stay (P = 0.0002). Multivariate analysis revealed male gender (odds ratio: 2.57, P = 0.0170), prior history of upper abdominal surgery (odds ratio: 5.53, P = 0.0132), and a higher preoperative white blood cell count (odds ratio: 3.62, P = 0.0244) to be independent factors associated with the requirement for an additional port. CONCLUSION: Our results suggest that gender, prior history of upper abdominal surgery, and white blood cell count can predict the likelihood of requiring an additional port in SILC.

Targeting hyaluronan for the treatment of pancreatic ductal adenocarcinoma.

Progression of cancer is often associated with interactions between cancer cells and extracellular matrix (ECM) surrounding them. Increasing evidence has suggested that accumulation of hyaluronan (HA), a major component of ECM, provides a favorable microenvironment for cancer progression. Pancreatic ductal adenocarcinoma (PDAC) is characterized typically by a dense desmoplastic stroma with a large amount of HA, making this molecule as an attractive target for therapy. Several studies have shown efficacy of inhibitors of HA synthesis or signaling for the treatment of PDAC. Recent studies have also demonstrated substantial improvements in the effects of chemotherapy by a targeted depletion of stromal HA in PDAC using an enzymatic agent. Thus, targeting HA has been recognized as a promising therapeutic strategy to treat this highly aggressive neoplasm. In this review article, we summarize our current understanding of the role of HA in the progression of PDAC and discuss possible therapeutic approaches targeting HA.

Hyaluromycin, a Novel Hyaluronidase Inhibitor, Attenuates Pancreatic Cancer Cell Migration and Proliferation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by accelerated production and degradation of hyaluronan (HA), a major component of extracellular matrix involved in the malignant phenotype of cancer. In particular, increased hyaluronidase (HYAL) activity plays a critical role in cancer progression, at least in part, by producing low-molecular-weight- (LMW-) HA or small fragments of HA, suggesting HYAL as a target for cancer treatment. Hyaluromycin, a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces hyaluromycini as a HYAL inhibitor. We investigated the antitumor effects of hyaluromycin in PDAC cells. We examined the effects of hyaluromycin on the proliferation and migration of PDAC cells. To elucidate the mechanisms underlying the effect of hyaluromycin on PDAC cells, we examined the concentration of LMW-HA in the conditioned media after treating PDAC cells with hyaluromycin. We demonstrate that hyaluromycin inhibits proliferation and migration of PDAC cells. We also found that these antitumor effects of hyaluromycin were associated with a decreased concentration of LMW-HA and a decreased phosphorylation of ribosomal protein S6. Our results suggest that hyaluromycin is a promising new drug against this highly aggressive neoplasm.

A novel epigenetic mechanism regulating hyaluronan production in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma enriched with hyaluronan (HA), a major component of extracellular matrix known to play a critical role in tumor progression. The mechanisms that regulate HA synthesis in PDAC are poorly understood. To investigate whether DNA methylation and HA production from PDAC cells are associated, we studied the effect of 5-aza-2'-deoxycitidine (5-aza-dC), an inhibitor of DNA methylation, or DNA methyltransferase 1 (DNMT1) knockdown by small interfering RNA, on the HA production from PDAC cells. HA production into the conditioned medium was evaluated in PDAC cells treated with 5-aza-dC or DNMT1 knockdown. mRNA expression of HA synthase (HAS) genes was investigated by real-time RT-PCR. Treatment of PDAC cells with 5-aza-dC led to a significant increase in the HA production (up to 2.5-fold increase) in all 4 cell lines tested. This enhanced HA production by 5-aza-dC treatment was accompanied by increased mRNA expression of HAS2 and HAS3. Furthermore, increased HA production and HAS2/HAS3 mRNA expression was also observed in PDAC cells by knockdown of DNMT1. These findings provide evidence, for the first time, that epigenetic mechanism is involved in the regulation of HA synthesis in PDAC cells.

Hyaluronan stimulates pancreatic cancer cell motility.

Hyaluronan (HA) accumulates in pancreatic ductal adenocarcinoma (PDAC), but functional significance of HA in the aggressive phenotype remains unknown. We used different models to investigate the effect of HA on PDAC cell motility by wound healing and transwell migration assay. Changes in cell motility were examined in 8 PDAC cell lines in response to inhibition of HA production by treatment with 4-methylumbelliferone (4-MU) and to promotion by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by co-culture with tumor-derived stromal fibroblasts. We also investigated changes in cell motility by adding exogenous HA. Additionally, mRNA expressions of hyaluronan synthases and hyaluronidases were examined using real time RT-PCR. Inhibition of HA by 4-MU significantly decreased the migration, whereas promotion of HA by TPA or co-culture with tumor-derived fibroblasts significantly increased the migration of PDAC cells. The changes in HA production by these treatments tended to be associated with changes in HAS3 mRNA expression. Furthermore, addition of exogenous HA, especially low-molecular-weight HA, significantly increased the migration of PDAC cells. These findings suggest that HA stimulates PDAC cell migration and thus represents an ideal therapeutic target to prevent invasion and metastasis.

Receptor for Hyaluronic Acid-Mediated Motility is Associated with Poor Survival in Pancreatic Ductal Adenocarcinoma.

Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) is a nonintegral cell surface receptor involved in the aggressive phenotype in a wide spectrum of human malignancies, but the significance of RHAMM in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, we investigated the expression of RHAMM and its clinical relevance in PDAC. RHAMM mRNA expression was examined in 8 PDAC cell lines and in primary pancreatic cancer and adjacent non-tumor tissues from 14 patients using real-time RT-PCR. Western blotting was carried out to analyze the expression of RHAMM protein in PDAC cell lines. We also investigated the expression patterns of RHAMM protein in tissue samples from 70 PDAC patients using immunohistochemistry. The RHAMM mRNA expression was increased in some PDAC cell lines as compared to a non-tumorous pancreatic epithelial cell line HPDE. The RHAMM mRNA expression was significantly higher in PDAC tissues as compared to corresponding non-tumorous pancreatic tissues (P < 0.0001). The RHAMM protein expression was higher in the vast majority of PDAC cell lines relative to the expression in HPDE. The immunohistochemical analysis revealed strong expression of RHAMM in 52 (74%) PDAC tissues. Strong expression of RHAMM was significantly associated with a shorter survival time (P = 0.038). In multivariate analysis, tumor stage (P = 0.039), residual tumor (P = 0.015), and strong RHAMM expression (P = 0.034) were independent factors predicting poor survival. Strong expression of RHAMM may predict poor survival in PDAC patients and may provide prognostic and, possibly, therapeutic value.