CD271 promotes proliferation and migration in bladder cancer.

Bladder cancer is a urothelial cancer and effective therapeutic strategies for its advanced stages are limited. Here, we report that CD271, a neurotrophin receptor, promotes the proliferation and migration of bladder cancer cells. CD271 knockdown decreased proliferation in both adherent and spheroid cultures, and vice versa when CD271 was overexpressed in bladder cancer cell lines. CD271 depletion impaired tumorigenicity in vivo. Migration activity was reduced by CD271 knockdown and TAT-Pep5, a known CD271-Rho GDI-binding inhibitor. Apoptosis was induced by CD271 knockdown. Comprehensive gene expression analysis revealed alterations in E2F- and Myc-related pathways upon CD271 expression. In clinical cases, patients with high CD271 expression showed significantly shortened overall survival. In surgically resected specimens, pERK, a known player in proliferation signaling, colocalizes with CD271. These data indicate that CD271 is involved in bladder cancer malignancy by promoting cell proliferation and migration, resulting in poor prognosis.

CD271 mRNA/hnRNPA2B1 complex promotes proliferation and stemness in oral and head and neck squamous cell carcinoma.

RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma.

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

Types of cell culture inserts affect cell crosstalk between co-cultured macrophages and adipocytes.

Cell culture inserts offer an in vivo-like microenvironment to investigate cell-cell interactions between co-cultivated cells. However, it is unclear if types of inserts affect cell crosstalk. Here, we developed an environment-friendly cell culture insert, XL-insert, which can reduce plastic waste with lower cost. We compared XL insert with two types of commercial disposable culture inserts, Koken® insert with atelocollagen membrane (Col-inserts) and Falcon® inserts with plastic membrane (PET-inserts) on cell-cell interactions in co-cultivated THP-1 macrophages and OP9 adipocytes. Scanning electron microscope, immunoassay and imaging analysis showed that among three types of inserts, XL-inserts allowed cytokines from co-cultivated macrophages and adipocytes to diffuse freely and offered preferable in vivo-like microenvironment for cell-cell interactions. PET-inserts showed limitations for intercellular communication due to some pores being blocked by somas on the membrane that caused much lower permeability for cytokines passing through. Col-inserts blocked large sized cytokines but allowed small sized molecules to permeate resulting in improved lipid accumulation and adiponectin secretion in OP9 adipocytes. Taken together, our data demonstrated that membrane type and pore size on the membrane affect the cross-talk between co-cultivated cells very differently. Some previous co-culture studies might have different results if the inserts were changed.

BEX2 is poor prognostic factor and required for cancer stemness in gastric cancer.

Gastric cancer is the fifth most common malignancy worldwide. However, targeted therapy for advanced gastric cancer is still limited. Here, we report BEX2 (Brain expressed X-linked 2) as a poor prognostic factor in two gastric cancer cohorts. BEX2 expression was increased in spheroid cells, and its knockdown decreased aldefluor activity and cisplatin resistance. BEX2 was found to upregulate CHRNB2 (Cholinergic Receptor Nicotinic Beta 2 Subunit) expression, a cancer stemness-related gene, in a transcriptional manner, and the knockdown of which also decreases aldefluor activity. Collectively, these data are suggestive of the role of BEX2 in the malignant process of gastric cancer, and as a promising therapeutic target.

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry.

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell-like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.

Discovery of a chemical compound that suppresses expression of BEX2, a dormant cancer stem cell-related protein.

Cancer stem cells (CSCs) are believed to cause cancer metastasis and recurrence. BEX2 (brain expressed X-linked gene 2) is a CSC-related gene that is expressed in dormant CSCs in cholangiocarcinoma and induces resistance against chemotherapy. The aim of the present study was to identify small compounds that have activity to inhibit BEX2 expression and result in the attenuation of CSC-related phenotypes. We screened 9600 small chemical compounds in high-throughput screening using cholangiocarcinoma cell line HuCCT1 expressing BEX2 protein fused with NanoLuc, and identified a compound, BMPP (1, 3-Benzenediol, [4-(4-methoxyphenyl)-1H-pyrazol-3-yl]). BMPP was found to exert decreasing effects on BEX2 protein expression and G0 phase population of the tumor cells, and increasing effects on ATP levels and chemotherapeutic sensitivity of the cells. These findings indicate that BMPP is a valuable chemical compound for reducing dormant CSC-related phenotypes. Thus, the identification of BMPP as a potential CSC suppressor provides scope for the development of novel therapeutic modalities for the treatment of cancers with BEX2 overexpressing CSCs.

Novel activating KRAS mutation candidates in lung adenocarcinoma.

Lung adenocarcinoma (LUAC) is a unique lung cancer subtype that is responsive to several therapeutic agents. The KRAS gene is the second most frequently mutated gene in LUAC and the majority of KRAS mutations are one of three classical activating mutations (G12, G13, and Q61). Recently, other types of "minor" KRAS mutation have been identified among LUAC patients and some may have similar transforming activities to those of the classical KRAS mutations. Here we describe minor KRAS mutations in LUAC patients, some of which (A66T, A66V, and G75E) may have tumor-forming activity in mouse embryonic fibroblasts in an allograft model. RNA-Seq analysis revealed that mouse embryonic fibroblasts overexpressing these three minor KRAS mutations have distinct expression profiles compared with overexpression of the wild type but similar expression profiles compared with overexpression of the classical KRAS mutants. Our results indicate that some of the minor KRAS mutations cause varying tumor formation activity and are important targets for developing anti-RAS agents as chemotherapeutic agents.

CD109 promotes the tumorigenic ability and metastatic motility of pancreatic ductal adenocarcinoma cells.

BACKGROUND: Accumulating evidence indicates that CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in human epithelial carcinomas of multiple organs including the pancreas, but its functional role in carcinoma development has not yet been fully clarified. The aim of this study was to investigate the role of CD109 in the malignancy of pancreatic ductal adenocarcinoma (PDAC). METHODS: PDAC specimens of 145 cases were immunostained for CD109, and correlations between CD109 expression and clinicopathological conditions were analyzed. CD109 expression in PANC-1 cells, a PDAC-derived cell line, was decreased by siRNA or shRNA and its effect on the malignancy of PANC-1 cells was examined. RESULTS: Suppression of CD109 expression in PANC-1 cells resulted in reduction of in vitro cell motility and tumorigenicity in xenografts. Based on these results, we investigated the relationship between CD109 expression and metastasis of PDAC using tumor tissue specimens. Among 106 recurrent cases of 145 PDAC, there was a tendency for CD109-positive cases to be accompanied by distant metastasis. CONCLUSIONS: CD109 plays a critical role in the promotion of tumorigenic ability and cellular motility relating to metastasis of PDAC cells.

CD271 is a negative prognostic factor and essential for cell proliferation in lung squamous cell carcinoma.

Squamous cell carcinoma is a major type of cancer in the lung. While several therapeutic target molecules for lung adenocarcinoma have been identified, little is known about lung squamous cell carcinoma (LSCC). We recently reported that CD271 (p75 neurotrophin receptor) serves as a marker for tumor initiation and is a key regulator of cell proliferation in hypopharyngeal squamous cell carcinoma. In this study, we found that CD271 was also expressed in squamous cell carcinoma, but not in adenocarcinoma, of several tissues, including the lung, and the expression of CD271 was associated with a poor prognosis in LSCC. To examine CD271's role in LSCC, we established xenograft cell lines from LSCC patients. Within the sorted live LSCC cell population, the CD271high cells were primarily cycling through the G2/M phase, while the CD271low cells were mostly in the G0 phase. CD271 knockdown in the LSCC cells completely suppressed their proliferation and tumor-formation capability, and increased their cell-cycle arrest in the G0 phase. In the CD271-knockdown cells, ERK-phosphorylation was decreased, while no change was observed in the IκBα-phosphorylation, p65-phosphorylation, or Akt-phosphorylation. Treatment with the MEK inhibitor U0126 decreased the LSCC cells' proliferation capability. Microarray analysis revealed that CD271 knockdown attenuated the RAS-related pathways. The knockdown of TrkB, which forms a heterodimer with CD271 and accelerates its downstream signaling, partially inhibited the LSCC cell proliferation. These results indicated that LSCC exclusively depends on CD271 for cell proliferation, in part through ERK-signaling activation, and CD271 is a promising target for LSCC therapy.

Maximization of calcium and phosphate in neonatal total parenteral nutrition.

BACKGROUND: Appropriate calcium and phosphate supplementation is essential for bone growth in preterm infants. Using Rehabix-K2™ (AY Pharmaceuticals, Tokyo, Japan) and Pleamin-P Injection™ (Fuso Pharmaceutical Industries, Osaka, Japan) as the total parenteral nutrition (TPN) and amino acid solution, respectively, we investigated ways of maximizing calcium and phosphate in the TPN solution. METHODS: Rehabix-K2, Pleamin-P, calcium gluconate, sodium phosphate, 50% glucose, and water were mixed in varying proportions to create 16 formulations. Precipitation assessment was done three times for each of the 16 formulations, and was based on the Japanese Pharmacopeia. RESULT: Precipitation was observed 24 h after mixing when the calcium and phosphate were 60 mEq/L and 30 mmol/L or 80 mEq/L and 40 mmol/L, respectively. No precipitation was observed when the calcium and phosphate were 20 mEq/L and 10 mmol/L, respectively. Precipitation was observed once out of three times, when the calcium and phosphate were 40 mEq/L and 20 mmol/L, respectively, and the amino acids were 2% and 3% (mean pH, 6.13 and 6.26, respectively). No precipitation was observed, however, when the calcium and phosphate were 40 mEq/L and 20 mmol/L, respectively, and the amino acids were 0% and 1% (mean pH, 5.88 and 6.05, respectively). CONCLUSION: Not only the concentration of calcium and phosphate, but also the pH of the TPN solution, are crucial factors for precipitation. Based on these results, a well-balanced TPN solution maximizing calcium and phosphate availability will be able to be formulated.

Biochemical characterization of thermostable ß-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus oryzae.

A ß-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant AoMan134A was expressed in Pichia pastoris, and the purified enzyme produced mannobiose, mannotriose, mannotetraose, and mannopentaose from galactose-free ß-mannan, with mannotriose being the predominant reaction product. The catalytic efficiency (k cat/K m ) of AoMan134A was 6.8-fold higher toward galactomannan from locust bean gum, than toward galactomannan from guar gum, but similar toward galactomannan from locust bean gum and glucomannan from konjac flour. After incubation at 70°C for 120 min, the activity of AoMan134A toward glucomannan decreased to 50% of the maximal activity at 30°C. AoMan134A retained 50% of its ß-1,4-mannanase activity after heating at 90°C for 30 min, indicating that AoMan134A is thermostable. Furthermore, AoMan134A was stable within a neutral-to-alkaline pH range, as well as exhibiting stability in the presence of a range of organic solvents, detergents, and metal ions. These findings suggest that AoMan134A could be useful in a diverse range of industries where conversion of ß-mannans is of prime importance.

Novel ß-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans.

Many filamentous fungi produce ß-mannan-degrading ß-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel ß-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free ß-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with ß-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-ß-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward ß-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when ß-mannans were the sole carbon source, indicating that Man134A is involved in ß-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of ß-mannan degradation that is enhanced by the novel ß-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-ß-1,4-mannanases.

Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells.

Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate.

Ethyl-2-hydroxy-4-methylpentanoate (ethyl leucate) contributes to a fruity flavor in Japanese sake. The mold Aspergillus oryzae synthesizes leucate from leucine and then the yeast Saccharomyces cerevisiae produces ethyl leucate from leucate during sake fermentation. Here, we investigated the enzyme involved in leucate synthesis by A. oryzae. The A. oryzae gene/cDNA encoding the enzyme involved in leucate synthesis was identified and expressed in E. coli and A. oryzae host cells. The purified recombinant enzyme belonged to a D-isomer-specific 2-hydroxyacid dehydrogenase family and it NADPH- or NADH-dependently reduced 4-methyl-2-oxopentanate (MOA), a possible intermediate in leucine synthesis, to D-leucate with a preference for NADPH. Thus, we designated this novel enzyme as MOA reductase A (MorA). Furthermore, an A. oryzae strain overexpressing morA produced 125-fold more leucate than the wild-type strain KBN8243. The strain overexpressing MorA produced 6.3-fold more ethyl leucate in the sake than the wild-type strain. These findings suggest that the strain overexpressing morA would help to ferment high-quality sake with an excellent flavor. This is the first study to identify the MOA reductase responsible for producing D-leucate in fungi.

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.

Synergistic cytotoxicity of afatinib and cetuximab against EGFR T790M involves Rab11-dependent EGFR recycling.

EGFR is an important therapeutic target for non-small cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, are effective in cases with EGFR-activating mutations. However, most such cases become resistant through a secondary EGFR mutation, T790M. While the second-generation TKI afatinib has a higher affinity for double-mutant EGFRs, better efficacy is needed. Combining afatinib with the anti-EGFR monoclonal antibody cetuximab improves clinical outcomes, but the mechanism is unclear. Here we examined this effect using erythroleukemic K562 cells. The activating EGFR mutation L858R is sensitive to first-generation TKIs, and adding T790M confers resistance to these drugs. This double-mutant EGFR was moderately sensitive to afatinib, but responded weakly to cetuximab. Combined afatinib and cetuximab synergistically increased their cytotoxicity for K562 cells expressing the double-mutant EGFR. Apoptosis in these cells followed induction of the pro-apoptotic protein BIM. Unexpectedly, afatinib caused redistribution of EGFR to the cell surface through Rab11a-dependent recycling. Cetuximab reduced cell-surface EGFR, and total EGFR decreased synergistically when cetuximab was combined with afatinib. Our results suggest that the synergistic effect exerted by afatinib and cetuximab on NSCLCs is associated with BIM induction and alterations in EGFR status.

Interplay of co-cultured chimeric adipose and gingival tissues exacerbates inflammatory dysfunction relevant to periodontal and metabolic conditions.

Adipose tissue dysfunction is a key feature of metabolic syndrome, which increases the risk of periodontitis, an inflammatory disease induced by bacteria that affects the gingiva and other components of periodontal tissue. Recent studies indicate that molecules from inflamed periodontal tissue contribute to adipose tissue dysfunction. However, the cellular mechanisms and interactions between adipose tissue and gingiva driving the progression of metabolic and periodontal conditions remain unclear. To address this, we developed a chimeric (mouse/human) co-culture tissue model (which identifies the origins of species-specific cytokines) to investigate these interactions. Using tissue-specific functional cells and immunocytes, we constructed equivalents of adipose tissue (ATE) and gingiva (GTE), co-cultivating them under inflammatory conditions induced by bacterial endotoxin, lipopolysaccharide (LPS). Our findings showed that exposure to LPS resulted in a notable reduction in lipid accumulation, GLUT4 expression, and adiponectin secretion in ATE, along with increased macrophage colonies forming around lipid droplets, as well as elevated levels of triglyceride, leptin, and IL-6. In GTE, LPS triggered significant inflammatory responses, characterized by increased macrophage accumulation, elevated COX-2 expression, and heightened secretion of inflammatory cytokines. LPS also reduced epithelial thickness and the expression of keratin 19 and collagen IV, indicating impaired barrier function and gingival integrity. Co-culturing ATE with GTE exacerbated these LPS-induced harmful effects in both tissues. In conclusion, our findings suggest that interplay between gingiva and adipose tissue can intensify the inflammatory and dysfunctional changes caused by LPS. This co-culture tissue model offers a valuable tool for future studies on periodontitis and metabolic syndrome.