The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 μM. Cell viability, cell cycling and direct interaction between GSK-3β and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3β and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed additivity at lower concentrations, but antagonism at higher concentrations. GSK-3β could directly phosphorylate 4EBP1 and activate the mTORC1 downstream signaling cascades to enhance protein biosynthesis and cell proliferation in RCC cell lines independent of rapamycin sensitivity. The direct GSK-3β/4EBP1 pathway might be an important subcellular mechanism as an inherent equipment for RCC cells to acquire clinical chemoresistance to mTORC1 inhibitors.

Background: The regulation of the redox balance in the tumor microenvironment is thought to be an adaptive response of tumor cells to hypoxic environments. In recent years, it has been reported that the hemoglobin β-chain (HBB), which is involved in scavenging reactive oxygen species (ROS), is expressed in several carcinomas. However, the relationship between HBB expression and the prognosis of renal cell carcinoma (RCC) remains unclear. Methods: HBB expression was immunohistochemically analyzed in 203 nonmetastatic clear cell RCC (ccRCC) cases. Cell proliferation, invasion, and ROS production were measured in ccRCC cell lines treated with HBB-specific siRNA. Results: The prognosis of HBB-positive patients was worse than that of HBB-negative patients. Cell proliferation and invasion were inhibited, and ROS production was increased by treatment with HBB-specific siRNA. Oxidative stress increased HBB expression in cells exposed to H₂O₂. Conclusions: HBB expression in ccRCC contributes to cancer cell proliferation by suppressing ROS production under hypoxic conditions. Taken together with clinical results and in vitro experiments, HBB expression may serve as a new prognostic biomarker for RCC in the future.

Solitary fibrous tumor (SFT) is a rare neoplasm composed of mesenchymal-derived spindle cells. Although SFT occurs anywhere in the body, they most frequently affects the thoracic region. Here, we reported an extremely rare case of an extrathoracic SFT occurring primarily in the kidney. To our knowledge, little information has been described on the immunohistochemistry (IHC) and genetics of renal SFT. A 41-year old Japanese female came to our hospital for further examination of a left kidney mass detected incidentally with ultrasound. Extensive investigation of the tumor, including physical, laboratory, and image examinations led to a clinical diagnosis of renal cancer (cT1aN0M0), which were in most parts imbedded in the lower polar parenchyma. The patient underwent laparoscopic radical nephrectomy. The mass was diagnosed pathologically as SFT originating from the kidney, but not as renal carcinoma. Microscopically, the tumor was composed of spindle-shape cells distributed variably in dense collagenous stroma and had a focal hemangiopericytomatous staghorn-like vascular pattern. Mitotic figures, atypical structures, necrosis and hemorrhage were not identified. No adjuvant therapies were given postoperatively. The patient has been free of tumor recurrence for 25 months since the surgery. IHC revealed that the tumor diffusely expressed CD34, CD99, Bcl2, PAX8, NAB2, STAT6, and GRIA2. The tumor stained negatively for desmin, S-100, c-Kit, CK-AE1/AE3, CDK4 and MDM2. A NAB2-SATA6 gene fusion was detected in tumor cells by reverse transcription-polymerase chain reaction, direct sequencing, and an in situ proximity ligation brightfield assay. The gene fusion occurred as an 831 bp truncation of exon 2 in NAB2 connected to the beginning of exon 3 in STAT6. We have reported a case of GRIA2 and PAX8-positive SFT occurring primarily in the kidney with such NAB2-STAT6 gene fusion for the first time. Diffuse expression of PAX8 in the tumor might present with a renal origin. Reportedly, benign histology of SFT cannot necessarily predict favorable clinical prognosis. Genetic alterations recently identified in SFT could possibly refer to risk stratification for tumor recurrence. However, malignant preponderance of extrathoracic SFT over thoracic SFT remains unexplained so far. Long-term follow-up after surgery should be performed in the present case.

Recent studies have discovered a relationship between glycosylphosphatidylinositol (GPI)-anchored protein 80 (GPI-80)/VNN2 (80 kDa GPI-anchored protein) and malignant tumors. GPI-80 is known to regulate neutrophil adhesion; however, the action of GPI-80 on tumors is still obscure. In this study, although the expression of GPI-80 mRNA was detectable in several tumor cell lines, the levels of GPI-80 protein were significantly lower than that in neutrophils. To clarify the function of GPI-80 in tumor cells, GPI-80-expressing cells and GPI-80/VNN2 gene-deleted cells were established using PC3 prostate cancer cells. In GPI-80-expressing cells, GPI-80 was mainly detected in vesicles. Furthermore, soluble GPI-80 in the conditioned medium was associated with the exosome marker CD63 and was also detected in the plasma obtained from prostate cancer patients. Unexpectedly, cell adhesion and migration of GPI-80-expressing PC3 cells were not modulated by anti-GPI-80 antibody treatment. However, similar to the GPI-80 family molecule, VNN1, the pantetheinase activity and oxidative state were augmented in GPI-80-expressing cells. GPI-80-expressing cells facilitated non-adhesive proliferation, slow cell proliferation, NF-κB activation and IL-1β production. These phenomena are known to be induced by physiological elevation of the oxidative state. Thus, these observations indicated that GPI-80 affects various tumor responses related to oxidation.

Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3‐deoxy‐dmanno‐octulosonic acid (dOclA) dimer and the N‐acetylglucosamine disaccharide of lipid A. X‐ray diffraction reveals that the type of cell is monoclinic (a = 5.53 Å, b = 27.2 Å, c = 6.47 Å , α = 90°, β = 125.8°, γ = 90°). Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b‐axis value, and two types of surface topographies are visualized. One, regarded as the view onto the acyl chain ends, is two‐dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a‐ and c‐axes and the angle of β (lattice I). The other, regarded as the view onto the dOclA dimers, is two‐dimensional arrays of dromedary‐back‐like bodies that constitute the lattice with axes of 9.0 and 10.7 Å and the angle of 65° formed by both axes (lattice II). Based on these results, we present the molecular model of E. coli Re LPS.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette–Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.