Pyruvate kinase type M2 contributes to the development of pancreatic ductal adenocarcinoma by regulating the production of metabolites and reactive oxygen species.

The majority of cancer cells maintain a high glycolytic activity and an increased lactate production, even in a well oxygenated environment. This phenomenon is known as the Warburg effect. Previous studies have revealed that various types of cancer selectively express the pyruvate kinase M2 isoform (PKM2), and that PKM2 plays a pivotal role in the Warburg effect. Although elevated PKM2 levels have been observed in pancreatic cancer and other types of cancer, little is known about the biological function of PKM2. In this study, in order to examine the expression and role of PKM2 in pancreatic ductal adenocarcinoma (PDAC), we knocked down PKM2 in PDAC cells by introducing small interfering and short hairpin RNAs, and examined the gene expression profiles in the cells by microarray analysis. We analyzed the energy-producing pathways in the cells by XFe Extracellular Flux Analyzers, and detected intracellular metabolites by capillary electrophoresis time-of-flight mass spectrometry. We found that the RNAi-mediated knockdown of PKM2 diminished the proliferative, migratory and tumorigenic ability of the PDAC cell-lines. PKM2 knockdown also resulted in lower glycolytic activities and decreased levels of some intracellular metabolites, such as pyruvate and polyamine; however, it led to elevated levels of reactive oxygen species. Microarray analysis revealed the functional association between PKM2 and the expression of genes that drive the cell cycle. On the whole, the findings of this study demonstrate that PKM2 plays an important role in metabolic activities, as well as in the malignancy of PDAC cells.

Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer-specific metabolism.

Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.

Enhanced expression of semaphorin 3E is involved in the gastric cancer development.

Semaphorins and their receptors are abnormally expressed in various cancers, but little is known about the expression and function of semaphorin 3E (SEMA3E) and its receptor, plexin D1 (PLXND1), in gastric cancer development or metastasis. We evaluated SEMA3E and PLXND1 expression by quantitative RT-PCR in gastric tissues from 62 patients who underwent gastrectomy and analyzed the correlation between their expression and clinicopathological variables. To assess the function of SEMA3E, we generated human gastric cancer cell lines with suppressed or increased SEMA3E expression. The expression level of SEMA3E, but not PLXND1, was correlated with lymph node involvement and metastatic progression in gastric cancer. A significant association was observed between a high level of SEMA3E expression and poor differentiation or poor survival in the intestinal type of gastric cancer. SEMA3E knockdown in gastric cancer cells attenuated cell proliferation and metastatic ability in vitro and in vivo. Moreover, SEMA3E caused cell proliferation and anchorage-independent cell growth in the intestinal type of gastric cancer. These results suggested that SEMA3E is likely to be involved in the development of gastric cancer and might also be a therapeutic target for its treatment.

Circulating miR-483-3p and miR-21 is highly expressed in plasma of pancreatic cancer.

Several recent studies have revealed that microRNAs (miRNAs) have a role in carcinogenesis and cancer development, and that it is stably detectable in plasma/serum. The aim of this study was to test whether miR­483-3p as well as miR­21 could be plasma biomarkers for PDAC. The plasma samples were obtained from three groups including 32 pancreatic ductal adenocarcinoma (PDAC) patients, 12 patients with intraductal papillary mucinous neoplasm (IPMN) patients and 30 healthy controls (HC). We evaluated the plasma miR­483-3p and miR­21 expression level by quantitative RT-PCR. We compared the differences in the plasma level of these miRNAs among the three groups, and investigated the relevance of their plasma expression level to the clinical factors in PDAC. The expressions of miR­483-3p and miR­21 were detected in all examined plasma samples. The plasma expression levels of these miRNAs were significantly higher in PDAC compared to HC (P<0.01). The plasma miR­483-3p expression was significantly higher in PDAC patients than IPMN patients (P<0.05). The plasma miR­21 level was associated with advanced stage (P<0.05), metastasis to lymph node and liver (P<0.01), and shorter survival (P<0.01) of the PDAC patients. Together, these findings suggest that measurement of the plasma miR­483-3p level is useful for discriminating PDAC from IPMN, and that the plasma miR­21 level predicts outcome of PDAC patients.

Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma.

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274(low) and CD274(high) cells from each cell line, and xenografted them into immunodeficient NOD/scid/γcnull (NOG) mice. We found that the CD274(low) cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274(high) cells. Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

Enhanced expression of long non-coding RNA HOTAIR is associated with the development of gastric cancer.

The long non-coding RNA HOTAIR has been reported to be a poor prognostic biomarker in a variety of malignant tumors. However, little is known about the association of HOTAIR with gastric cancer. We examined the expression of HOTAIR in 68 gastric cancer samples using quantitative real-time RT-PCR and analyzed its correlation with the clinical parameters. The functional role of HOTAIR was examined by generating human gastric cancer cell lines with increased or suppressed HOTAIR expression. The anchorage -independent growth was assessed by soft agar assay. The increased or suppressed HOTAIR expressing gastric cancer cells were injected into the tail vein or peritoneal cavity of immunodeficient mice to examine the effect of this molecule on metastasis and peritoneal dissemination. The expression of HOTAIR was significantly higher in cancer lesions than in adjacent non-cancerous tissues in human gastric cancers. In the diffuse type of gastric cancer, the High-HOTAIR group (HOTAIR/GAPDH > 1) showed significantly more venous invasion, frequent lymph node metastases and a lower overall survival rate compared to the Low-HOTAIR group (HOTAIR/GAPDH < 1). Colony formation on the soft agar was enhanced in a HOTAIR-dependent manner. HOTAIR-expressing MKN74 formed more liver metastasis compared to control when they were injected into the tail vein of mice. In addition, reduced expression of HOTAIR in KATO III suppressed peritoneal dissemination. These results suggest that HOTAIR plays a pivotal role in the development of gastric cancer.

Large noncoding RNA HOTAIR enhances aggressive biological behavior and is associated with short disease-free survival in human non-small cell lung cancer.

HOTAIR is one of long non-coding RNAs and its expression correlates with the prognosis and metastasis in various cancers. We showed that HOTAIR expression has an important role in the development of non-small cell lung cancer (NSCLC). In this study, we examined the expression of HOTAIR in 77 NSCLCs, their corresponding normal lung tissues and 6 brain metastases by quantitative real-time RT-PCR. High expression of HOTAIR (tumor/normal ratio ⩾2) was detected in 17 patients (22.1%) and was frequently found in patients with advanced stage, lymph node metastasis or lymph-vascular invasion and short disease free interval. Furthermore, brain metastases show significantly higher HOTAIR expression compared to primary cancer tissues. HOTAIR-expressing A549 cells showed induced cell migration and anchorage-independent cell growth in vitro. These results indicate the expression of HOTAIR enhanced the aggressive behavior of NSCLC cells.