The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.

Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture.

The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.

Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB.

Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.

The interferon-ß/STAT1 axis drives the collective invasion of skin squamous cell carcinoma with sealed intercellular spaces.

The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-ß was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-ß was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.

Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, nonnatural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.

Matrix Stiffness Contributes to Cancer Progression by Regulating Transcription Factors.

Matrix stiffness is critical for the progression of various types of cancers. In solid cancers such as mammary and pancreatic cancers, tumors often contain abnormally stiff tissues, mainly caused by stiff extracellular matrices due to accumulation, contraction, and crosslinking. Stiff extracellular matrices trigger mechanotransduction, the conversion of mechanical cues such as stiffness of the matrix to biochemical signaling in the cells, and as a result determine the cellular phenotypes of cancer and stromal cells in tumors. Transcription factors are key molecules for these processes, as they respond to matrix stiffness and are crucial for cellular behaviors. The Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is one of the most studied transcription factors that is regulated by matrix stiffness. The YAP/TAZ are activated by a stiff matrix and promotes malignant phenotypes in cancer and stromal cells, including cancer-associated fibroblasts. In addition, other transcription factors such as ß-catenin and nuclear factor kappa B (NF-κB) also play key roles in mechanotransduction in cancer tissues. In this review, the mechanisms of stiffening cancer tissues are introduced, and the transcription factors regulated by matrix stiffness in cancer and stromal cells and their roles in cancer progression are shown.

Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

Glycans, including glycosphingolipids, are broadly expressed in plasma membranes and play important roles in cell-cell interactions. Recently, it has been revealed that glycans participate in the regulation of malignant phenotypes of cancer cells, e.g. growth and invasion. However, their roles in irradiation-tolerant cancer cells have not yet been elucidated. In this study, we show that specific glycosphingolipids are highly expressed in invasive, irradiation-tolerant lung cancer cells. Particularly, the glycosphingolipid GM2 contributes to the development of an invasive phenotype in these lung cancer cells. Our results suggest that glycosphingolipids, including GM2, are implicated in the regulation of invasiveness in irradiation-tolerant lung cancer cells and may therefore serve as potential therapeutic targets for lung cancers following radiotherapy.Key words: glycosphingolipids, GM2, invasion, lung cancer cells, radiotherapy.

Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells.

In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179-89. ©2017 AACR.

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.

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.

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.

Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells.

Radiotherapy is effective for treating various types of tumors. However, some cancer cells survive after irradiation and repopulate tumors with highly malignant phenotypes that correlate with poor prognosis. It is not known how cancer cells survive and generate malignant tumors after irradiation. Here, we show that activating transcription factor 5 (ATF5) promotes radioresistance and malignancy in cancer cells after irradiation. In the G1-S phase of the cell cycle, cancer cells express high levels of ATF5, which promotes cell cycle progression and thereby increases radioresistance. Furthermore, ATF5 increases malignant phenotypes, such as cell growth and invasiveness, in cancer cells in vitro and in vivo. We have identified a new mechanism for the regeneration of highly malignant tumors after irradiation and shown that ATF5 plays a key role in the process.

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 ß1, 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.

Lung cancer cells that survive ionizing radiation show increased integrin α2ß1- 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 ß1 subunits were significantly elevated in IR cells. Knockdown of α2 expression or functional blockade of integrin α2ß1 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 α2ß1 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.

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.