TSPAN12 is a critical factor for cancer-fibroblast cell contact-mediated cancer invasion.

Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non-cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the ß-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced ß-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy.

Shear stress-induced redistribution of vascular endothelial-protein-tyrosine phosphatase (VE-PTP) in endothelial cells and its role in cell elongation.

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.

Preparation of Chemically Resistant Cellulose Benzoate Hollow Fiber Membrane via Thermally Induced Phase Separation Method.

For the first time, we have successfully fabricated microfiltration (MF) hollow fiber membranes by the thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS) methods using cellulose acetate benzoate (CBzOH), which is a cellulose derivative with considerable chemical resistance. To obtain an appropriate CBzOH TIPS membrane, a comprehensive solvent screening was performed to choose the appropriate solvent to obtain a membrane with a porous structure. In parallel, the CBzOH membrane was prepared by the NIPS method to compare and evaluate the effect of membrane structure using the same polymer material. Prepared CBzOH membrane by TIPS method showed high porosity, pore size around 100 nm or larger and high pure water permeability (PWP) with slightly low rection performance compared to that by NIPS. On the contrary, CBzOH membranes prepared with the NIPS method showed three times lower PWP with higher rejection. The chemical resistance of the prepared CBzOH membranes was compared with that of cellulose triacetate (CTA) hollow fiber membrane, which is a typical cellulose derivative as a control membrane, using a 2000 ppm sodium hypochlorite (NaClO) solution. CBzOH membranes prepared with TIPS and NIPS methods showed considerable resistance against the NaClO solution regardless of the membrane structure, porosity and pore size. On the other hand, when the CTA membrane, as the control membrane, was subjected to the NaClO solution, membrane mechanical strength sharply decreased over the exposure time to NaClO. It is interesting that although the CBzOH TIPS membrane showed three times higher pure water permeability than other membranes with slightly lower rejection and considerably higher NaClO resistance, the mechanical strength of this membrane is more than two times higher than other membranes. While CBzOH samples showed no change in chemical structure and contact angle, CTA showed considerable change in chemical structure and a sharp decrease in contact angle after treatment with NaClO. Thus, CBzOH TIPS hollow fiber membrane is noticeably interesting considering membrane performance in terms of filtration performance, mechanical strength and chemical resistance on the cost of slightly losing rejection performance.

Preparation of Microfiltration Hollow Fiber Membranes from Cellulose Triacetate by Thermally Induced Phase Separation.

For the first time, self-standing microfiltration (MF) hollow fiber membranes were prepared from cellulose triacetate (CTA) via the thermally induced phase separation (TIPS) method. The resultant membranes were compared with counterparts prepared from cellulose diacetate (CDA) and cellulose acetate propionate (CAP). Extensive solvent screening by considering the Hansen solubility parameters of the polymer and solvent, the polymer's solubility at high temperature, solidification of the polymer solution at low temperature, viscosity, and processability of the polymeric solution, is the most challenging issue for cellulose membrane preparation. Different phase separation mechanisms were identified for CTA, CDA, and CAP polymer solutions prepared using the screened solvents for membrane preparation. CTA solutions in binary organic solvents possessed the appropriate properties for membrane preparation via liquid-liquid phase separation, followed by a solid-liquid phase separation (polymer crystallization) mechanism. For the prepared CTA hollow fiber membranes, the maximum stress was 3-5 times higher than those of the CDA and CAP membranes. The temperature gap between the cloud point and crystallization onset in the polymer solution plays a crucial role in membrane formation. All of the CTA, CDA, and CAP membranes had a very porous bulk structure with a pore size of ∼100 nm or larger, as well as pores several hundred nanometers in size at the inner surface. Using an air gap distance of 0 mm, the appropriate organic solvents mixed in an optimized ratio, and a solvent for cellulose derivatives as the quench bath media, it was possible to obtain a CTA MF hollow fiber membrane with high pure water permeance and notably high rejection of 100 nm silica nanoparticles. It is expected that these membranes can play a great role in pharmaceutical separation.

TSPAN2 is involved in cell invasion and motility during lung cancer progression.

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transition and that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, a transmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis to the lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species.

NuMA is required for the selective induction of p53 target genes.

The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

Requirement of ATM for rapid p53 phosphorylation at Ser46 without Ser/Thr-Gln sequences.

p53 phosphorylation at Ser46 following DNA damage is important for preferential transactivation of proapoptotic genes. Here, we report that ataxia-telangiectasia mutated (ATM) kinase is responsible for Ser46 phosphorylation of p53 during early-phase response to DNA damage. To elucidate the direct phosphorylation of p53 at Ser46 by ATM, an ATM mutant (ATM-AS) sensitive to ATP analogues was engineered. In vitro kinase assays revealed that p53 was phosphorylated at Ser46 by ATM-AS, even when ATP analogues were used as phosphate donors, although this phosphorylation site is not in an SQ motif, a consensus ATM site. Furthermore, Ser46 phosphorylation by ATM was dependent on the N- and C-terminal domains of p53, unlike Ser15 phosphorylation. Immunofluorescence analyses showed that Ser46-phosphorylated p53 was observed as foci in response to DNA damage and colocalized with gamma-H2AX or Ser1981-phosphorylated ATM. These results suggest that ATM phosphorylates a noncanonical serine residue on p53 by mechanisms different from those for the phosphorylation of Ser15.