Comparative evaluation of round window niche accessibility pre-operatively on high-resolution computed tomography of the temporal bone with intra-operative findings.

OBJECTIVE: To compare round window niche visibility as seen endoscopically during cochlear implant surgery with pre-operative high-resolution computed tomography of the temporal bone. METHODS: Nineteen patients scheduled for cochlear implantation, aged 2-20 years, were referred for computed tomography from October 2016 to March 2018. Angles were measured between the lines passing through the mid-sagittal plane and cochlear basal turn on the scans. Endoscopic round window niche visibility during posterior tympanotomy was categorised as: type I = 100 per cent, type IIa = more than 50 per cent, type IIb = less than 50 per cent or type III = 0 per cent. Pre-operative computed tomography measurements were used to predict round window niche visibility before surgery and correlated with intra-operative findings. RESULTS: The mean (range) of pre-operative angles on computed tomography for endoscopic visibility types I, IIa and IIb, were 64.06° (61.16-69.37°), 63.81° (58.61-71.35°) and 56.48° (50.37-59.05°), respectively, a statistically significant finding (one-way analysis of variance test, p = 0.016). CONCLUSION: Pre-operative high-resolution temporal bone computed tomography measurements are useful in predicting round window niche visualisation as viewed endoscopically during posterior tympanotomy. The angle was more acute in type IIb compared to type I.

MUC1-C integrates PD-L1 induction with repression of immune effectors in non-small-cell lung cancer.

Immunotherapeutic approaches, particularly programmed death 1/programmed death ligand 1 (PD-1/PD-L1) blockade, have improved the treatment of non-small-cell lung cancer (NSCLC), supporting the premise that evasion of immune destruction is of importance for NSCLC progression. However, the signals responsible for upregulation of PD-L1 in NSCLC cells and whether they are integrated with the regulation of other immune-related genes are not known. Mucin 1 (MUC1) is aberrantly overexpressed in NSCLC, activates the nuclear factor-κB (NF-κB) p65→︀ZEB1 pathway and confers a poor prognosis. The present studies demonstrate that MUC1-C activates PD-L1 expression in NSCLC cells. We show that MUC1-C increases NF-κB p65 occupancy on the CD274/PD-L1 promoter and thereby drives CD274 transcription. Moreover, we demonstrate that MUC1-C-induced activation of NF-κB→︀ZEB1 signaling represses the TLR9 (toll-like receptor 9), IFNG, MCP-1 (monocyte chemoattractant protein-1) and GM-CSF genes, and that this signature is associated with decreases in overall survival. In concert with these results, targeting MUC1-C in NSCLC tumors suppresses PD-L1 and induces these effectors of innate and adaptive immunity. These findings support a previously unrecognized central role for MUC1-C in integrating PD-L1 activation with suppression of immune effectors and poor clinical outcome.

Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells.

Patients with HER2-positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane mucin 1 (MUC1) oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1 C-terminal subunit (MUC1-C) in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in the downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1-C/HER2 complexes and decreases in tyrosine-phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab-resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (∼20-fold) as compared with that in sensitive cells. In addition, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with the downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance.

Relationship between tumor enhancement, edema, IDH1 mutational status, MGMT promoter methylation, and survival in glioblastoma.

BACKGROUND AND PURPOSE: Both IDH1 mutation and MGMT promoter methylation are associated with longer survival. We investigated the ability of imaging correlates to serve as noninvasive biomarkers for these molecularly defined GBM subtypes. MATERIALS AND METHODS: MR imaging from 202 patients with GBM was retrospectively assessed for nonenhancing tumor and edema among other imaging features. IDH1 mutational and MGMT promoter methylation status were determined by DNA sequencing and methylation-specific PCR, respectively. Overall survival was determined by using a multivariate Cox model and the Kaplan-Meier method with a log rank test. A logistic regression model followed by ROC analysis was used to classify the IDH1 mutation and methylation status by using imaging features. RESULTS: MGMT promoter methylation and IDH1 mutation were associated with longer median survival. Edema levels stratified survival for methylated but not unmethylated tumors. Median survival for methylated tumors with little/no edema was 2476 days (95% CI, 795), compared with 586 days (95% CI, 507-654) for unmethylated tumors or tumors with edema. All IDH1 mutant tumors were nCET positive, and most (11/14, 79%) were located in the frontal lobe. Imaging features including larger tumor size and nCET could be used to determine IDH1 mutational status with 97.5% accuracy, but poorly predicted MGMT promoter methylation. CONCLUSIONS: Imaging features are potentially predictive of IDH1 mutational status but were poorly correlated with MGMT promoter methylation. Edema stratifies survival in MGMT promoter methylated but not in unmethylated tumors; patients with methylated tumors with little or no edema have particularly long survival.

Differential gene expression in glioblastoma defined by ADC histogram analysis: relationship to extracellular matrix molecules and survival.

BACKGROUND AND PURPOSE: ADC histogram analysis can stratify outcomes in patients with GBM treated with bevacizumab. Therefore, we compared gene expression between high-versus-low ADC tumors to identify gene expression modules that could underlie this difference and impact patient prognosis. MATERIALS AND METHODS: Up-front bevacizumab-treated patients (N = 38) with newly diagnosed glioblastoma were analyzed by using an ADC histogram approach based on enhancing tumor. Using microarrays, we compared gene expression in high-versus-low ADC tumors in patients subsequently treated with bevacizumab. Tissue sections from a subset of tumors were stained for collagen and collagen-binding proteins. Progression-free and overall survival was determined by using Cox proportional hazard ratios and the Kaplan-Meier method with the log rank test. RESULTS: A total of 13 genes were expressed at 2-fold or greater levels in high- compared with low-ADC tumors at the P < .05 level. Of these, 6 encode for collagen or collagen-binding proteins. High gene expression for the collagen-binding protein decorin was associated with shorter survival (HR, 2.5; P = .03). The pattern and degree of collagen staining were highly variable in both high- and low-ADC tumors. CONCLUSIONS: High-ADC GBMs show greater levels of ECM protein gene expression compared with low-ADC GBMs. It is unclear whether this translates to the accumulation of higher levels of the encoded proteins. However, because ECM molecules could contribute to a proinvasive phenotype, this relationship merits further investigation.

Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria.

Tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We generated cell lines from glioblastoma specimens with the goal to obtain model systems for glioma stem cell biology. Unsupervised analysis of the expression profiles of nine cell lines established under neural stem cell conditions yielded two distinct clusters. Four cell lines were characterized by the expression of neurodevelopmental genes. They showed a multipotent differentiation profile along neuronal, astroglial and oligodendroglial lineages, grew spherically in vitro, expressed CD133 and formed highly invasive tumors in vivo. The other five cell lines shared expression signatures enriched for extracellular matrix-related genes, had a more restricted differentiation capacity, contained no or fewer CD133+ cells, grew semiadherent or adherent in vitro and displayed reduced tumorigenicity and invasion in vivo. Our findings show that stable, multipotent glioblastoma cell lines with a full stem-like phenotype express neurodevelopmental genes as a distinctive feature, which may offer therapeutic targeting opportunities. The generation of another distinct cluster of cell lines showing similarly homogeneous profiling but restricted stem cell properties suggests that different phenotypes exist, each of which may lead to the typical appearance of glioblastoma.

Targeting of protein kinase C delta to mitochondria in the oxidative stress response.

The cellular response to oxidative stress includes the release of mitochondrial cytochrome c and the induction of apoptosis. Here we show that treatment of diverse cells with hydrogen peroxide (H2O2) induces the targeting of protein kinase C delta (PKCdelta) to mitochondria. The results demonstrate that H2O2-induced activation of PKCdelta is necessary for translocation of PKCdelta from the cytoplasm to the mitochondria. The results also show that mitochondrial targeting of PKCdelta is associated with the loss of mitochondrial transmembrane potential and release of cytochrome c. The functional importance of this event is also supported by the demonstration that H2O2-induced apoptosis is blocked by the inhibition of PKCdelta activation and translocation to mitochondria. These findings indicate that mitochondrial targeting of PKCdelta is required, at least in part, for the apoptotic response of cells to oxidative stress.

Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells.

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of the stress-activated protein kinase (SAPK) and induction of terminal monocytic differentiation. The present studies demonstrate that TPA targets SAPK to mitochondria by a mechanism dependent on activation of protein kinase C (PKC) beta. Translocation of SAPK to mitochondria in response to TPA is associated with release of cytochrome c, caspase-3 activation and induction of apoptosis. The results show that TPA induces the association of SAPK with the mitochondrial anti-apoptotic Bcl-x(L) protein. Overexpression of Bcl-x(L) attenuated the apoptotic response to TPA treatment. Moreover, expression of Bcl-x(L) mutated at sites of SAPK phosphorylation (Thr-47, -115) was more effective than wild-type Bcl-x(L) in abrogating TPA-induced cytochrome c release and apoptosis. By contrast, expression of Bcl-x(L) had little effect on induction of the monocytic phenotype. These findings indicate that myeloid leukemia cells respond to TPA with targeting of SAPK to mitochondria and that this response contributes to terminal differentiation through the release of cytochrome c and induction of apoptosis.

Targeting of the c-Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress-induced apoptosis.

The ubiquitously expressed c-Abl tyrosine kinase localizes to the nucleus and cytoplasm. Using confocal microscopy, we demonstrated that c-Abl colocalizes with the endoplasmic reticulum (ER)-associated protein grp78. Expression of c-Abl in the ER was confirmed by immunoelectron microscopy. Subcellular fractionation studies further indicate that over 20% of cellular c-Abl is detectable in the ER. The results also demonstrate that induction of ER stress with calcium ionophore A23187, brefeldin A, or tunicamycin is associated with translocation of ER-associated c-Abl to mitochondria. In concert with targeting of c-Abl to mitochondria, cytochrome c is released in the response to ER stress by a c-Abl-dependent mechanism, and ER stress-induced apoptosis is attenuated in c-Abl-deficient cells. These findings indicate that c-Abl is involved in signaling from the ER to mitochondria and thereby the apoptotic response to ER stress.

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Apaf-1/cytochrome c-independent and Smac-dependent induction of apoptosis in multiple myeloma (MM) cells.

Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c (cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced caspase-9 activation and apoptosis in MM cells.

Targeting of the c-Abl tyrosine kinase to mitochondria in the necrotic cell death response to oxidative stress.

The ubiquitously expressed c-Abl tyrosine kinase is activated in the response of cells to genotoxic and oxidative stress. The present study demonstrates that reactive oxygen species (ROS) induce targeting of c-Abl to mitochondria. We show that ROS-induced localization of c-Abl to mitochondria is dependent on activation of protein kinase C (PKC)delta and the c-Abl kinase function. Targeting of c-Abl to mitochondria is associated with ROS-induced loss of mitochondrial transmembrane potential. The results also demonstrate that c-Abl is necessary for ROS-induced depletion of ATP and the activation of a necrosis-like cell death. These findings indicate that the c-Abl kinase targets to mitochondria in response to oxidative stress and thereby mediates mitochondrial dysfunction and cell death.

The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces monocytic differentiation of human myeloid leukemia cells and inhibits proliferation of diverse human tumor cell lines. The present studies on human osteosarcoma cells demonstrate that CDDO induces mitochondrial cytochrome c release, caspase-3 activation, and internucleosomal DNA fragmentation. Overexpression of the caspase-8 inhibitor CrmA blocked CDDO-induced cytochrome c release and apoptosis. By contrast, overexpression of the antiapoptotic Bcl-x(L) protein blocked CDDO-induced cytochrome c release, but only partly inhibited caspase-3 activation and apoptosis. In concert with these findings, we demonstrate that CDDO: 1) activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism and 2) induces cytochrome c release by caspase-8-dependent cleavage of Bid. The results also demonstrate that treatment of osteosarcoma cells with CDDO induces differentiation, as assessed by alkaline phosphatase activity and osteocalcin production, and that this response is abrogated in cells that overexpress CrmA. These findings demonstrate that CDDO induces both osteoblastic differentiation and apoptosis by caspase-8-dependent mechanisms.

The ARG tyrosine kinase interacts with Siva-1 in the apoptotic response to oxidative stress.

The Abl family of mammalian nonreceptor tyrosine kinases consists of c-Abl and ARG (Abl-related gene). Certain insights are available regarding the involvement c-Abl in the response of cells to stress. ARG, however, has no known function in cell signaling. The present studies demonstrate that ARG associates with the proapoptotic Siva-1 protein. The functional significance of the ARG-Siva-1 interaction is supported by the finding that ARG is activated by oxidative stress and that this response involves ARG-mediated phosphorylation of Siva-1 on Tyr(48). The proapoptotic effects of Siva-1 are accentuated in cells stably expressing ARG and are inhibited in ARG-deficient cells. Moreover, the proapoptotic effects of Siva-1 are abrogated by mutation of the Tyr(48) site. We also show that the apoptotic response to oxidative stress is attenuated in ARG-deficient cells and that this defect is corrected by reconstituting ARG expression. These findings support a model in which the activation of ARG by oxidative stress induces apoptosis by a Siva-1-dependent mechanism.

Interaction of hematopoietic progenitor kinase 1 and c-Abl tyrosine kinase in response to genotoxic stress.

The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The hematopoietic progenitor kinase 1 (HPK1) has also been shown to act upstream to the SAPK/JNK signaling pathway. We report here that exposure of hematopoietic Jurkat T cells to genotoxic agents is associated with activation of HPK1. The results demonstrate that exposure of Jurkat cells to DNA-damaging agents is associated with translocation of active c-Abl from nuclei to cytoplasm and binding of c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks c-Abl-mediated induction of SAPK/JNK. These findings indicate that activation of HPK1 and formation of HPK1/c-Abl complexes are functionally important in the stress response of hematopoietic cells to genotoxic agents.

Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin.

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.

The novel isocoumarin 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) induces lethality of human carcinoma cells by generation of reactive oxygen species.

2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) is an isocoumarin derivative that has recently entered clinical trials for evaluation as a p.o.-bioavailable, antiangiogenic molecule. NM-3 induces endothelial cell death at low microM concentrations by a nonapoptotic mechanism. The present studies have assessed the direct effects of NM-3 on human carcinoma cells. The results demonstrate that NM-3 treatment is associated with the generation of reactive oxygen species and loss of clonogenic survival. In concert with these findings, we show that exposure to NM-3 is associated with increases in expression of the p53 tumor suppressor. In human MCF-7 and ZR-75-1 breast cancer cells, NM-3 induces the p21 cyclin-dependent kinase inhibitor, cell cycle arrest at G1-S-phase, and necrotic cell death. Moreover, human PA-1 ovarian carcinoma and HeLa cervical carcinoma cells respond to NM-3 with the induction of apoptosis by a reactive oxygen species-dependent mechanism. These findings demonstrate that NM-3 has direct effects on carcinoma cells at clinically achievable concentrations and that this agent could be effective in targeting both the tumor and its vasculature.

BAL is a novel risk-related gene in diffuse large B-cell lymphomas that enhances cellular migration.

Clinical risk factor models such as the International Prognostic Index are used to identify diffuse large B-cell lymphoma (DLB-CL) patients with different risks of death from their diseases. To elucidate the molecular bases for these observed clinical differences in outcome, differential display was used to identify a novel gene, termed BAL (B-aggressive lymphoma), which is expressed at significantly higher levels in fatal high-risk DLB-CLs than in cured low-risk tumors. The major BAL complementary DNA encodes a previously uncharacterized 88-kd nuclear protein with a duplicated N-terminal domain homologous to the nonhistone portion of histone-macroH2A and a C-terminal alpha-helical region with 2 short coiled-coil domains. Of note, the BAL N-terminus and secondary structure resemble those of a recently identified human protein, KIAA1268. In addition, both BAL and KIAA1268 map to chromosome 3q21, further suggesting that these genes belong to a newly identified family. BAL is expressed at increased levels in DLB-CL cell lines with an activated peripheral B cell, rather than a germinal center B cell, phenotype. This observation and the characteristic dissemination of high risk DLB-CLs prompted studies regarding the role of BAL in B-cell migration. In classical transwell assays, stable BAL-overexpressing B-cell lymphoma transfectants had significantly higher rates of migration than vector-only transfectants, indicating that the risk-related BAL gene promotes malignant B-cell migration. (Blood. 2000;96:4328-4334)

Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90.

The release of cytochrome c from mitochondria results in the formation of an Apaf-1-caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90-Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.