Yoshihiro Yui

Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers.

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD‐fmk did not completely rescue FK228‐induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule‐associated protein light chain 3 (LC3‐I) to conjugated light chain 3 (LC3‐II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase‐independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228‐induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228‐induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl+) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8

The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin β1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin β1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.

Changes in cell migration of mesenchymal cells during osteogenic differentiation

We showed that the migration, morphology and adhesiveness of undifferentiated mesenchymal cells dramatically changed during osteogenic differentiation. The migration of these cells was transiently upregulated early in osteogenic differentiation. At a later stage, migration was decreased but adhesiveness was increased. Furthermore, Cdc42 and Rac1 Rho‐family small GTPases were activated at early stages of differentiation and the phosphorylation level of FAK decreased as differentiation progressed. We also showed cell migration was promoted by inhibition of the Rho‐ROCK‐myosin signaling. Finally, using a mouse model of ectopic bone formation, we confirmed that treatment with ROCK inhibitor, Y‐27632 increased cell movement into bone formation sites, resulting in enhanced osteogenesis. These results provide a new insight into the link between cell migration and osteogenic differentiation.

Involvement of Extracellular Signal-Regulated Kinase Activation in Human Osteosarcoma Cell Resistance to the Histone Deacetylase Inhibitor FK228 [(1S,4S,7Z,10S,16E,21R)-7-Ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone]

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC50) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury.

Successful autologous peripheral blood stem cell transplantation with a double-conditioning regimen for recurrent hepatoblastoma after liver transplantation

Abstract:  A four‐yr‐old boy developed a solitary metastasis nine months after living‐related liver transplantation for unresectable hepatoblastoma. After resection of the metastatic lesion, he received an auto‐PBSCT with a double‐conditioning regimen consisting of melphalan and thiotepa. Auto‐PBSCT could be safely performed without any serious regimen‐related toxicity or infection. However, transient cessation of tacrolimus during myelosuppression resulted in graft rejection of the liver just after hematological engraftment, but rejection was resolved by tacrolimus and methylprednisolone. The patient is alive and free from disease two yr after auto‐PBSCT without any signs of graft rejection. High‐dose chemotherapy using this conditioning regimen may be feasible for recurrent hepatoblastoma after liver transplantation in terms of safety and anti‐tumor activity.

A pediatric case of transfusion‐related acute lung injury following bone marrow infusion

Abstract:  TRALI is a rare but serious complication associated with transfusion, and known to occur following infusion of all types of plasma‐containing blood products. However, only one adult case of TRALI after allogenic marrow graft has been reported. In this study, we present a pediatric case possibly associated with allogenic marrow infusion. A 10‐yr‐old girl was referred to our hospital for the treatment of acute myeloid leukemia. She underwent allogenic bone marrow transplantation from her HLA‐2‐loci‐mismatched mother. During conditioning, she suffered from bacterial sepsis, but it had improved with antibiotics until day 0 of transplantation. Two h after starting the marrow infusion, she developed severe hypoxia. We discontinued the infusion and started steroids, which improved her respiratory condition. However, she developed respiratory failure again after resuming infusion of the graft. Despite intensive care with mechanical ventilation, the patient died of endotoxin shock five days after transplantation. Although we could not identify the antibody which might have been involved in the respiratory distress, the clear temporal relationship between marrow infusion and respiratory distress suggested that similar acute lung injury to TRALI might have occurred following allogenic marrow infusion in the present case.

Abstract PR09: Extracellular stiffness cues drive spatial reorganization of the genome to globally constrain RNA abundance

The stiffness of the extracellular matrix (ECM) drives mechanosignaling that regulates tissue development and malignancy. We previously showed that a stiff ECM disrupts tissue organization and enhances malignant progression by inducing cell invasion and migration. However, the specific transcriptional and molecular events in which mechanotransduction directs these phenotypes are not well understood. To clarify this process, we used a combination of genome-scale approaches to monitor changes in gene expression and protein abundance as a function of acinar morphogenesis and tissue homeostasis in three dimensional extracellular matrix hydrogels with tunable stiffness. Elevated ECM stiffness perturbed tissue homeostasis and reverted the transcriptional phenotype of differentiated mammary acini to resemble that observed in rapidly proliferating nonpolarized mammary cell aggregates. These findings suggest that tissue tension induces cellular changes that directly reflect higher-order tissue organizational states. We found that these changes involve the spatial rearrangement of peripheral chromatin, and that the expression levels of multiple histone deacetylases increase in organized tissues concurrently with elevated nuclear heterochromatin content, an effect that is abrogated in rigid ECM conditions. We support these observations by mapping mechanoresponsive peripheral heterochromatin elements via ChIPseq, enabling us to directly identify dynamic regions containing genes whose transcriptional activity is responsive to mechanical cues. Finally, using a combination of genomic, imaging, and molecular biology techniques we demonstrated that ECM compliance and tissue organization significantly influences global RNA abundance. Notably, this model presents formidable conceptual and practical challenges for the interpretation of genomic data. Collectively, this work indicates that tissue organization is critically dependent on the cellular mechanical environment, which qualitatively and quantitatively shapes the epigenetic and transcriptional landscape by mechanisms that have not yet been elucidated. Note: This abstract was not presented at the conference. Citation Format: Russell Bainer, Yoshihiro Yui, Shannon Mumenthaler, Parag Mallick, Lin Liu, Hua-Jun Wu, Ondrej Podlaha, Franziska Michor, Jan Liphardt, Jonathan Licht, Valerie Weaver. Extracellular stiffness cues drive spatial reorganization of the genome to globally constrain RNA abundance. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR09.