Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis.

Dose-related cardiomyopathy is a major side effect following doxorubicin (Dox). To investigate whether exercise (Ex)-induced vasculogenesis plays a role in reducing Dox-induced cardiotoxicity, GFP+ bone marrow (BM) cells from GFP transgenic mice were transplanted into wild-type mice. Transplanted mice were treated with Dox, Ex, Dox+Ex, or control. We found Dox therapy resulted in decreased systolic and diastolic blood flow, decreased ejection fraction and fractional shortening, and decreased vascular endothelial cells and pericytes. These abnormalities were not seen in Dox+Ex hearts. Heart tissues from control-, Ex-, or Dox-treated mice showed a small number of GFP+ cells. By contrast, the Dox+Ex-treated hearts had a significant increase in GFP+ cells. Further analyses demonstrated these GFP+ BM cells had differentiated into vascular endothelial cells (GFP+CD31+) and pericytes (GFP+NG2+). Decreased cardiomyocytes were also seen in Dox-treated but not Dox+Ex-treated hearts. Ex induced an increase in GFP+c-Kit+ cells. However, these c-Kit+ BM stem cells had not differentiated into cardiomyocytes. Dox therapy induced phosphorylation of MST1/2, LATS1, and YAP; a decrease in total YAP; and cleavage of caspase-3 and PARP in the heart tissues. Dox+Ex prevented these effects. Our data demonstrated Dox-induced cardiotoxicity is mediated by vascular damage resulting in decreased cardiac blood flow and through activation of Hippo-YAP signaling resulting in cardiomyocyte apoptosis. Furthermore, Ex inhibited these effects by promoting migration of BM stem cells into the heart to repair the cardiac vessels damaged by Dox and through inhibiting Dox-induced Hippo-YAP signaling-mediated apoptosis. These data support the concept of using exercise as an intervention to decrease Dox-induced cardiotoxicity.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Transcriptional repressor REST drives lineage stage-specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-ß (infants) subgroups. Neuronal maturation is greater in SHH-ß than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG ) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1 +/- cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context-specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1 Expression of Arrb1, which encodes ß-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed RESTTG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1 These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-ß than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation.

The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma.Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073-84. ©2017 AACR.

Medulloblastoma development: tumor biology informs treatment decisions.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatments including surgery, craniospinal radiation and high-dose chemotherapy have led to improvement in survival. However, the risk for recurrence as well as significant long-term neurocognitive and endocrine sequelae associated with current treatment modalities underscore the urgent need for novel tumor-specific, normal brain-sparing therapies. It has also provided the impetus for research focused on providing a better understanding of medulloblastoma biology. The expectation is that such studies will lead to the identification of new therapeutic targets and eventually to an increase in personalized treatment approaches.

HuR Suppresses Fas Expression and Correlates with Patient Outcome in Liver Cancer.

UNLABELLED: Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3'-untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3'-UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties. IMPLICATIONS: Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

The peptide derived from the Ig-like domain of human herpesvirus 8 K1 protein induces death in hematological cancer cells.

BACKGROUND: Although significant progress has been made in the treatment of lymphomas, many lymphomas exhibit resistance to cell death, suggesting a defective Fas signaling, which remains poorly understood. We previously reported that cells expressing the K1 protein of human herpesvirus 8 (HHV-8) resist death through the complex formation of the Ig-like domain of K1 with Fas. Recently, we investigated whether peptides derived from the Ig-like domain of the K1 protein may affect cell death. METHODS: K1 positive and negative cell lines were incubated with the K1-derived peptides, and cell death (apoptotic and necrotic) was assessed by flow cytometry and LDH assay. Activation of caspases was assessed by fluorometric assay and flow cytometry. Fas receptor-independent, peptide-mediated cell killing was tested in the Fas-resistant Daudi cell line and Jurkat cell clones deficient in caspase-8 and FADD functionality. Activation of TNF receptors I and II was blocked by pre-incubation with corresponding blocking antibodies. The effect of the K1 peptide in vivo was tested in a mouse xenograft model. RESULTS: We observed that the peptide S20-3 enhanced cell death in K1-positive BJAB cells and HHV-8 positive primary effusion lymphoma (PEL) cell lines. Similar effects of this peptide were observed in B-cell lymphoma and T-lymphoblastic leukemia cells without K1 expression but not in normal human peripheral blood mononuclear cells. A single intratumoral injection of the S20-3 peptide decreased the growth of Jurkat xenografts in SCID mice. The mechanism of tumor cell death induced by the S20-3 peptide was associated with activation of caspases, but this activity was only partially inhibited by the pan-caspase inhibitor z-VAD. Furthermore, the K1 peptide also killed Fas-resistant Daudi cells, and this killing effect was inhibited by pre-incubation of cells with antibodies blocking TNFRI. CONCLUSION: Taken together, these findings indicate that the S20-3 peptide can selectively induce the death of malignant hematological cell lines by Fas- and/or TNFRI-dependent mechanisms, suggesting the K1-derived peptide or peptidomimetic may have promising therapeutic potential for the treatment of hematological cancers.

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo.

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

Milatuzumab - a promising new immunotherapeutic agent.

Milatuzumab is a new immunotherapeutic agent targeting CD74, a membrane protein preferentially expressed in hematopoietic cancers and some solid tumors. Broad expression and fast internalization makes CD74 an ideal target for cancer therapy. We reviewed published articles about CD74 and milatuzumab. We present a comprehensive review of CD74 functions and provide explanation of milatuzumab antitumor effects. This review describes CD74 protein biology with the emphasis on the role of CD74 in tumor survival and its new role in regulation of the Fas death receptor. The development of CD74 targeting therapies to induce tumor regression and cancer cell apoptosis is described and results of clinical trials are discussed. Milatuzumab shows selective binding and rapid internalization into CD74-positive cancer cells. Milatuzumab with and without conjugated toxins synergizes with other chemotherapeutic agents and elicits significant antitumor effects in mice. In a Phase I trial, milatuzumab showed no severe adverse effects in patients with relapsed/refractory multiple myeloma and it stabilized the disease in some patients for up to 12 weeks. Ongoing trials testing different treatment schedules of milatuzumab in chronic lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma indicate that milatuzumab shows no severe adverse effects in humans.

All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB or HER, plays crucial roles in the development of multicellular organisms. Mutations and over-expression of the ErbB receptors have been implicated in a variety of human cancers. It is widely thought that the ErbB receptors are located in the plasma membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, it still remains controversial whether prior to ligand binding the receptors exist as monomers or dimers on the cell surface. Using bimolecular fluorescence complementation (BiFC) assays in the present study, we demonstrate that in the absence of bound ligand, all the ErbB family members have preformed, yet inactive, homo- and heterodimers on the cell surface, except for ErbB3 homodimers and heterodimers with cleavable ErbB4, which exist primarily in the nucleus. BiFC assays of the dimerization have also suggested that the ligand-independent dimerization of the ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Based on BiFC and mammalian two-hybrid assays, it is apparent that the intracellular domains of the receptors are responsible for the spontaneous dimer formation. These provide new insights into an understanding of transmembrane signal transduction mediated by the ErbB family members, and are relevant to the development of anti-cancer drugs.

Opposite effects of alternative TZF spliced variants on androgen receptor.

We previously demonstrated that testicular zinc-finger protein (TZF) was a corepressor of the androgen receptor (AR). In the present study, we further showed that TZF-L, an alternative spliced variant of TZF, enhanced transactivation function of AR. Deletion analysis of TZF-L revealed that its N-terminus, which almost corresponded to that of TZF, but not its C-terminus was able to interact with AR. Additional analysis suggested that TZF and TZF-L were able to form both homodimers and heterodimers. TZF-L inhibited the homodimer formation of TZF and the intranuclear dot formation of TZF. We propose that in the unique regulation system of AR-mediated transactivation, two spliced isoforms of TZF act as coactivator and corepressor, respectively.

Testicular zinc finger protein recruits histone deacetylase 2 and suppresses the transactivation function and intranuclear foci formation of agonist-bound androgen receptor competitively with TIF2.

We previously reported that testicular zinc finger protein (TZF) is a corepressor for androgen receptor (AR). The present study demonstrated that a central portion (amino acids 512-663) of TZF, TZF(512-663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512-663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.

Impaired nuclear translocation, nuclear matrix targeting, and intranuclear mobility of mutant androgen receptors carrying amino acid substitutions in the deoxyribonucleic acid-binding domain derived from androgen insensitivity syndrome patients.

CONTEXT: Recent imaging studies revealed that androgen receptor (AR) is ligand-dependently translocated from the cytoplasm into the nucleus and forms intranuclear fine foci. In this study, we examined whether intracellular dynamics of mutant ARs detected in two androgen insensitivity syndrome (AIS) patients was impaired. OBJECTIVE: ARs with mutations in the DNA-binding domain were functionally characterized and compared with the wild-type AR. PATIENTS: In a complete AIS patient (subject 1), cysteine residue 579 in the first zinc finger motif of AR was substituted for phenylalanine (AR-C579F). Another mutation (AR-F582Y) was found in a partial AIS patient (subject 2). RESULTS: AR-F582Y retained less than 10% of the transactivation activity of the wild-type AR, whereas no ligand-dependent transactivation was detected for AR-C579F. Image analyses of the receptors fused to green fluorescent protein showed that the wild-type AR was ligand-dependently translocated into the nucleus in which it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone, the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a part of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots, compared with the wild-type AR. Fluorescence recovery after photobleaching analysis revealed that the intranuclear mobility of the mutant ARs decreased, compared with that of the wild-type AR. CONCLUSIONS: These results suggest that the abnormal translocation, localization, and mobility of the mutant ARs may be the cause of AIS in these subjects.

A zinc finger protein TZF is a novel corepressor of androgen receptor.

Steroid hormones control the transcriptional activity of target genes mediated by intracellular nuclear receptors, and these transcriptional activities are modulated by the combination with coactivators and corepressors. We found in this study that testicular zinc finger protein (TZF) that was a nuclear protein with a zinc finger motif of the Cys2-His2 type was a novel corepressor of androgen receptor (AR). Fusion protein with green fluorescence protein GFP formed the specific foci in nuclei and TZF-dependent foci were located close to the splicing factor compartment. In addition, TZF was recruited into AR subnuclear foci after the treatment of dihydrotestosterone. Furthermore, we revealed that TZF bound to the activation function-1 (AF-1) domain (N-terminal transactivating domain) of AR protein. Transient over-expression of TZF in COS-7 cells or LNCaP human prostatic cancer cell resulted in decreased AR activity in a ligand-dependent fashion. Moreover, a transcriptional corepressor N-CoR additively decreased the transcriptional activity of AR with TZF. These findings suggest that TZF might be a novel corepressor of AR.