Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer.

Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

Calcitriol exerts an anti-tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response.

Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)-approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti-proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti-osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.

Synergistic antiproliferative effect of imatinib and adriamycin in platelet-derived growth factor receptor-expressing osteosarcoma cells.

Osteosarcoma (OS) is the most frequent primary solid malignant tumor of bone. Its prognosis remains poor in the substantial proportion of patients who do not respond to chemotherapy and novel therapeutic options are therefore needed. We previously established a mouse model that mimics the aggressive behavior of human OS. Enzyme-linked immunosorbent assay-based screening of such mouse tumor lysates identified platelet-derived growth factor-BB (PDGF-BB) as an abundant soluble factor, the gene for which was expressed dominantly in surrounding non-malignant cells of the tumor, whereas that for the cognate receptor (PDGF receptor ß) was highly expressed in OS cells. Platelet-derived growth factor-BB induced activation of both MEK-ERK and phosphatidylinositol 3-kinase-protein kinase B signaling pathways and promoted survival in OS cells deprived of serum, and these effects were blocked by the PDGF receptor inhibitor imatinib. However, these actions of PDGF-BB and imatinib were mostly masked in the presence of serum. Whereas imatinib alone did not manifest an antitumor effect in mice harboring OS tumors, combined treatment with imatinib and adriamycin exerted a synergistic antiproliferative effect on OS cells in vivo. These results suggest that treatment of OS with imatinib is effective only when cell survival is dependent on PDGF signaling or when imatinib is combined with another therapeutic intervention that renders the tumor cells susceptible to imatinib action, such as by inducing cellular stress.

IGF1 receptor signaling regulates adaptive radioprotection in glioma stem cells.

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular-signal-regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection.

Single­agent nintedanib suppresses metastatic osteosarcoma growth by inhibiting tumor vascular formation.

New therapeutic approaches are needed for osteosarcoma, which is the most common malignancy of the bone, especially for metastatic cases. Nintedanib is a potent, oral tyrosine kinase inhibitor approved for treating idiopathic pulmonary fibrosis, which blocks a variety of receptor signals, including fibroblast growth factor receptors, vascular endothelial growth factor receptors and platelet-derived growth factor receptors. The present study assessed the effect of nintedanib on previously developed mouse AXT osteosarcoma cells, and on AXT-derived osteosarcoma developed in C57BL/6 mice, which displays lethal tumors with osteoid formation and lung metastatic lesions that mimics human disease. In vitro analysis, including flow cytometry and immunoblotting, revealed that nintedanib inhibited AXT cell proliferation and cell cycle progression, induced apoptosis, and inactivated AKT and ERK1/2. Immunoblot analysis using tumor lysates demonstrated that nintedanib inhibited its target molecules in vivo. As a single agent, nintedanib decreased the size of primary AXT-derived osteosarcoma, and reduced circulating tumor cells and lung metastasis. Immunohistochemical findings indicated that nintedanib exerted antitumor activity mainly by inhibiting the formation of CD31-positive tumor vasculature, while αSMA-positive cells were still enriched in tumors after nintedanib treatment. In addition, nintedanib exhibited an anti-osteosarcoma effect on C57BL/6 severe combined immunodeficient mice in which T- and B-cell function is obsolete, suggesting that the antitumor effect of nintedanib was not attributable to antitumor immunity. Collectively, these findings indicated that nintedanib holds potential for treating osteosarcoma.

Keratinocyte-Derived Cytokine in the Hippocampus Disrupts Extinction of Conditioned Fear Memory in Tumor-Bearing Mice.

While patients with cancer show a higher prevalence of psychiatric disorders than the general population, the mechanism underlying this interaction remains unclear. The present study examined whether tumor-bearing (TB) mice show psychological changes using the conditioned fear paradigm and the role of cytokines in these changes. TB mice were established by transplantation with mouse osteosarcoma AXT cells. These TB mice were then found to exhibit disruption in extinction of conditioned fear memory. Eighteen cytokines in serum were increased in TB mice, among which i.c.v. injection of interleukin (IL)-1ß and IL-6 strengthened fear memory in normal mice. Contents of IL-17 and keratinocyte-derived cytokine (KC) in the amygdala and KC in the hippocampus were increased in TB mice. KC mRNA in both the amygdala and hippocampus was also increased in TB mice, and i.c.v. injection of KC dose-dependently strengthened fear memory in normal mice. In addition, injection of IL-1ß, but not IL-6, increased KC mRNA in the amygdala and hippocampus. In TB mice KC mRNA was increased in both astrocytes and microglia of the amygdala and hippocampus. The microglia inhibitor minocycline, but not the astrocyte inhibitor fluorocitrate, alleviated disruption in extinction of conditioned fear memory in TB mice. Microinjection of KC into the hippocampus, but not into the amygdala, increased fear memory in normal mice. These findings indicate that TB mice show an increase in serum cytokines, including IL-1ß, that increases KC production in microglia of the hippocampus, which then disrupts extinction of fear memory.

Potential risk of driving performance under combined conditions of taking second-generation antihistamines and attending calls using a hands-free function.

OBJECTIVE: Although second-generation antihistamines have reduced sedation-related side effects compared to first-generation antihistamines, sedation may still impair motor vehicle driving performance. Moreover, receiving/making phone calls using a hands-free function can negatively affect driving performance. Therefore, herein, driving performance was evaluated using a driving simulator to gain insights into the hazards of driving by combining second-generation antihistamines and a calling task, i.e., simulated calls using a hands-free function. METHODS: In this study, 20 subjects drove in a driving simulator in the absence or presence of a calling task while taking or not taking second-generation antihistamines. Driving performances for nonemergency and emergency events were determined, and a comparative analysis of intra-individual variability when taking and not taking second-generation antihistamines was conducted. RESULTS: First, when nonemergency and emergency were examined in the absence of a calling task, no significant difference in driving performance was observed between taking and not taking second-generation antihistamines. Next, when the nonemergency event was examined in the presence of a calling task, no significant difference in driving performance was observed between taking and not taking second-generation antihistamines. However, when the emergency event was examined in the presence of a calling task, a significant difference in driving performance was observed between taking and not taking second-generation antihistamines, thus resulting in reduced driving performance. CONCLUSIONS: The new system with added calling tasks allowed the extraction of the potential risks of driving performance of second-generation antihistamines that may have been previously overlooked. This study suggests that pharmacists and other healthcare professionals may need to instruct people taking any second-generation antihistamine to focus on driving and not on subtasks that require cognitive load such as talking while driving.

Usefulness of driver's eye movement measurement to detect potential risks under combined conditions of taking second-generation antihistamines and calling tasks.

BACKGROUND: Concerns persist regarding the potential reduction in driving performance due to taking second-generation antihistamines or performing hands-free calling. Previous studies have indicated a potential risk to driving performance under an emergency event when these two factors are combined, whereas a non-emergency event was operated effectively. Currently, there is a lack of a discriminative index capable of detecting the potential risks of driving performance impairment. This study aims to investigate the relationship between driving performance and eye movements under combined conditions of taking second-generation antihistamines and a calling task, and to assess the usefulness of eye movement measurements as a discriminative index for detecting potential risks of driving performance impairment. METHODS: Participants engaged in a simulated driving task, which included a calling task, both under taking or not taking second-generation antihistamines. Driving performance and eye movements were monitored during both emergency and non-emergency events, assessing their correlation between driving performance and eye movements. The study further evaluated the usefulness of eye movement as a discriminative index for potential driving impairment risk through receiver operating characteristic (ROC) analysis. RESULTS: In the case of a non-emergency event, no correlation was observed between driving performance and eye movement under the combined conditions. Conversely, a correlation was observed during an emergency event. The ROC analysis, conducted to assess the discriminative index capability of eye movements in detecting the potential risk of driving performance impairment, demonstrated a high discriminative power, with an area under the curve of 0.833. CONCLUSIONS: The findings of this study show the correlation between driving performance and eye movements under the concurrent influence of second-generation antihistamines and a calling task, suggesting the usefulness of eye movement measurement as a discriminant index for detecting potential risks of driving performance impairment.

Twist2 functions as a tumor suppressor in murine osteosarcoma cells.

The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.

Cancer aggravation due to persistent pain signals with the increased expression of pain-related mediators in sensory neurons of tumor-bearing mice.

A growing body of evidence suggests that intractable pain reduces both the quality of life and survival in cancer patients. In the present study, we evaluated whether chronic pain stimuli could directly affect cancer pathology using tumor-bearing mice. For this purpose, we used two different models of chronic pain in mice, neuropathic pain and persistent postsurgical pain, with Lewis lung carcinoma (LLC) as tumor cells. We found that tumor growth was dramatically promoted in these pain models. As well as these pain models, tumor growth of LLC, severe osteosarcoma (AXT) and B16 melanoma cells was significantly promoted by concomitant activation of sensory neurons in AAV6-hM3Dq-injected mice treated with the designer drug clozapine-N-oxide (CNO). Significant increases in mRNA levels of vascular endothelial growth factor-A (Vegfa), tachykinin precursor 1 (Tac1) and calcitonin-related polypeptide alpha (Calca) in the ipsilateral side of dorsal root ganglion of AAV6-hM3Dq-injected mice were observed by concomitant activation of sensory neurons due to CNO administration. Moreover, in a model of bone cancer pain in which mice were implanted with AXT cells into the right femoral bone marrow cavity, the survival period was significantly prolonged by repeated inhibition of sensory neurons of AAV6-hM4Di-injected mice by CNO administration. These findings suggest that persistent pain signals may promote tumor growth by the increased expression of sensory-located peptides and growth factors, and controlling cancer pain may prolong cancer survival.

Establishment of a choriocarcinoma model from immortalized normal extravillous trophoblast cells transduced with HRASV12.

Gestational choriocarcinoma is a malignant trophoblastic tumor. The development of novel molecular-targeted therapies is needed to reduce the toxicity of current multiagent chemotherapy and to treat successfully the chemoresistant cases. The molecular mechanisms underlying choriocarcinoma tumorigenesis remain uncharacterized, however, and appropriate choriocarcinoma animal models have not yet been developed. In this study, we established a choriocarcinoma model by inoculating mice with induced-choriocarcinoma cell-1 (iC³-1) cells, generated from HTR8/SVneo human trophoblastic cells retrovirally transduced with activated H-RAS (HRASV12). The iC³-1 cells exhibited constitutive activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and developed into lethal tumors in all inoculated mice. Histopathological analysis revealed that the tumors consisted of two distinct types of cells, reminiscent of syncytiotrophoblasts and cytotrophoblasts, as seen in the human choriocarcinoma. The tumors expressed HLA-G and cytokeratin (trophoblast markers) and hCG (a choriocarcinoma marker). Comparative analysis of gene expression profiles between iC³-1 cells and parental HTR8/SVneo cells revealed that iC³-1 cells expressed matrix metalloproteinases, epithelial-mesenchymal transition-related genes, and SOX3 at higher levels than parental trophoblastic cells. Administration of SOX3-specific short-hairpin RNA decreased SOX3 expression and attenuated the tumorigenic activity of iC³-1 cells, suggesting that SOX3 overexpression might be critically involved in the pathogenesis of choriocarcinoma. Our murine model represents a potent new tool for studying the pathogenesis and treatment of choriocarcinoma.

Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo.

Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway.

CD47 belongs to the immunoglobulin superfamily and is associated with ß-integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B-chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47-mediated apoptosis in human lymphoid malignant cells. Treatment with S-S diabody alone induced apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of S-S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK-1 cells. In gene expression profiling of the S-S diabody-treated MOLT-4 cells, hypoxia inducible factor (HIF)-1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF-1α, RTP801 and BNIP3 were increased. Knockdown of HIF-1α by siRNA repressed S-S diabody-induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S-S diabody might have potential as a novel therapeutic agent for B-CLL.

MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.

The Inhibitor of Apoptosis Protein Livin Confers Resistance to Fas-Mediated Immune Cytotoxicity in Refractory Lymphoma.

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg.

ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells.

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells.

Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity.

Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma. Mol Cancer Ther; 16(1); 182-92. ©2016 AACR.

HTLV-I Tax induces and associates with Crk-associated substrate lymphocyte type (Cas-L).

Crk-associated substrate lymphocyte type (Cas-L) is a docking protein that is heavily tyrosine phosphorylated by the engagement of beta1 integrins in T cells. In the present study, we attempted to evaluate the role of Cas-L in the pathophysiology of adult T-cell leukemia (ATL). Examination of peripheral blood mononuclear cells from ATL patients as well as ATL-derived T cell lines showed an elevation of Cas-L in these cells. We showed that tyrosine phosphorylation as well as expression of Cas-L was markedly elevated through the induction of human T-lymphotropic virus type I (HTLV-I) Tax in JPX-9 cells, with these cells showing marked motile behavior on the ligands for integrins. We next performed yeast two-hybrid screening of cDNA library from an HTLV-I-transformed T cell line, which resulted in the identification of Tax as a putative binding partner for Cas-L. Co-precipitation experiments revealed that the serine-rich region of Cas-L might serve as the binding site with the highest affinity for Tax. Co-localization study showed that Tax and Cas-L partly merged in the cytoplasm. Finally, we showed that exogenous Cas-L inhibited Tax-mediated transactivation of nuclear factor kappaB (NF-kappaB), while Tax-independent activation of NF-kappaB remained intact, hence indicating that Cas-L might specifically regulate Tax-NF-kappaB pathway.

[Persistent neutrophilia occurring after pneumonia: a differential diagnosis of neutrophilia based on the WHO classification].

We experienced a 85-year-old female patient with granulocytosis, which occurred after the bacterial pneumonia. The white blood cell counts remained high between 30,000/microl and 120,000/microl for around one year. As the serum G-CSF level was within the normal range and there were no tumors on CT scan images, the existence of G-CSF-producing solid tumors was unlikely. Bone marrow examination revealed hypercellularity without excess of blasts and hiatus leukemia, accompanied by mild dysplasia in myeloid cells and megakaryocytes. No chromosomal abnormalities in bone marrow samples were seen with G-banding and multi-color FISH methods. Major/minor BCR-ABL fusion genes were negative by RT-PCR. As previously reported by several investigators, we often experience difficulties in distinguishing atypical CML from CNL and CMML. In this report, we discussed how to diagnose the cause of granulocytosis based on a literature review.