Seiichiro Ishihara

Lung Cancer Cells That Survive Ionizing Radiation Show Increased Integrin α2β1- and EGFR-Dependent Invasiveness

Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin α2 and β1 subunits were significantly elevated in IR cells. Knockdown of α2 expression or functional blockade of integrin α2β1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule’s essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin α2β1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy.

Substrate stiffness regulates temporary NF-κB activation via actomyosin contractions

Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-κB, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness. NF-κB activation was temporarily induced in H1299 lung adenocarcinoma cells grown on a stiff substrate but not in cells grown on a soft substrate. Although the activation of NF-κB was independent of the activity of integrin β1, an ECM-binding protein, the activation was dependent on actomyosin contractions induced by phosphorylation of myosin regulatory light chain (MRLC). Additionally, the inhibition of MRLC phosphorylation by Rho kinase inhibitor Y27632 reduced the activity of NF-κB. We also observed substrate-specific morphology of the cells, with cells grown on the soft substrate appearing more rounded and cells grown on the stiff substrate appearing more spread out. Inhibiting NF-κB activation caused a reversal of these morphologies on both substrates. These results suggest that substrate stiffness regulates NF-κB activity via actomyosin contractions, resulting in morphological changes.

Integrin β1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta1-dependent phenotype, and integrin beta1 might be a potentially effective therapeutic target in combination with radiotherapy.

Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway

Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

Irradiation-tolerant lung cancer cells acquire invasive ability dependent on dephosphorylation of the myosin regulatory light chain

Radiotherapy is one of the major treatment modalities for malignancies. However, cells surviving irradiation often display high levels of invasiveness. This study shows that irradiation‐tolerant lung adenocarcinoma demonstrates high invasive capability depending on dephosphorylation of the myosin regulatory light chain (MRLC). In a collagen gel overlay condition, low‐invasive subclones of lung adenocarcinoma (A549P‐3) showed a round morphology and diphosphorylation of MRLC. In contrast, irradiation‐tolerant A549P‐3 cells (A549P‐3IR) displayed high invasiveness and a lower level of MRLC diphosphorylation. In addition, inhibition of MRLC phosphatase activity decreased the invasive activity. These findings suggest that A549P‐3IR cells acquire high invasiveness through MRLC dephosphorylation.

Filamin B enhances the invasiveness of cancer cells into 3D collagen matrices

Numerous types of cancer cells migrate into extracellular tissues. This phenomenon is termed invasion, and is associated with poor prognosis in cancer patients. In this study, we demonstrated that filamin B (FLNb), an actin-binding protein, is highly expressed in cancer cell lines that exhibit high invasiveness, with a spindle morphology, into 3D collagen matrices. In addition, we determined that knockdown of FLNb in invasive cancer cells converts cell morphology from spindle-shaped, which is associated with high invasiveness, to round-shaped with low invasiveness. Furthermore, di-phosphorylation of myosin regulatory light chain (MRLC) and phosphorylation of focal adhesion kinase (FAK) are inhibited in FLNb-knockdown cancer cells. These results suggest that FLNb enhances invasion of cancer cells through phosphorylation of MRLC and FAK. Therefore, FLNb may be a new therapeutic target for invasive cancers.

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.