HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

Mucin-like 1 (MUCL1) was first identified as a breast-specific gene over a decade ago. Based on its highly restricted mRNA expression in breast tissue and continued expression during breast tumorigenesis and progression, MUCL1 is an attractive tumor-associated antigen and a potential therapeutic target. However, very little is known about the cellular location, biological functions and regulation of the MUCL1 protein, which will have a major impact on its druggability. Here we describe our efforts to fully characterize the cellular localization of MUCL1, investigate its regulation by key breast cancer oncogenes such as human epidermal growth factor receptor 2 (HER2) and discover its functional roles in breast cancer. Although some mucins are membrane bound, our data indicate that MUCL1 is secreted by some breast cancer cells, whereas others only express high levels of intracellular MUCL1. MUCL1 expression is highest in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer.

Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors.

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.

Searching for targets for the systemic therapy of mesothelioma.

Malignant mesothelioma is an incurable disease associated with asbestos exposure arising in the pleural cavity and less frequently in the peritoneal cavity. Platinum-based combination chemotherapy with pemetrexed is the established standard of care. Multimodality approaches including surgery and radiotherapy are being investigated. Increasing knowledge about the molecular characteristics of mesothelioma had led to the identification of novel potential targets for systemic therapy. Current evidence suggests pathways activated in response to merlin deficiency, including Pi3K/mTOR and the focal adhesion kinase, as well as immunotherapeutic approaches to be most promising. This review elaborates on the rationale behind targeted approaches that have been and are undergoing exploration in mesothelioma and summarizes available clinical results and ongoing efforts to improve the systemic therapy of mesothelioma.

CD74: a new prognostic factor for patients with malignant pleural mesothelioma.

BACKGROUND: The pro-inflammatory cytokine migration inhibitory factor (MIF) and its receptor CD74 have been proposed as possible therapeutic targets in several cancers. We studied the expression of MIF and CD74 together with calretinin in specimens of malignant pleural mesothelioma (MPM), correlating their expression levels with clinico-pathologic parameters, in particular overall survival (OS). METHODS: Migration inhibitory factor, CD74, and calretinin immunoreactivity were investigated in a tissue microarray of 352 patients diagnosed with MPM. Protein expression intensities were semiquantitatively scored in the tumour cells and in the peritumoral stroma. Markers were matched with OS, age, gender, and histological subtype. RESULTS: Clinical data from 135 patients were available. Tumour cell expressions of MIF and CD74 were observed in 95% and 98% of MPM specimens, respectively, with a homogenous distribution between the different histotypes. CD74 (P<0.001) but not MIF overexpression (P=0.231) emerged as an independent prognostic factor for prolonged OS. High expression of tumour cell calretinin correlated with the epithelioid histotype and was also predictive of longer OS (P<0.001). When compared with previously characterised putative epithelial-to-mesenchymal transition markers, CD74 correlated positively with tumoral PTEN and podoplanin expressions, but was inversely related with periostin expression. CONCLUSIONS: High expression of CD74 is an independent prognostic factor for prolonged OS in mesothelioma patients.

Pain-related middle-latency somatosensory evoked potentials in the prognosis of post anoxic coma: a preliminary report.

BACKGROUND: Regarding hypoxic-ischemic encephalopathy, while the bilateral absence of N20/P25 somatosensory evoked potentials (SEPs) is considered to be the best indicator of adverse outcomes, the presence of middle latency evoked potentials (MLCEPs) is associated with a favourable neurological prognosis. The main aim of the present study was to investigate whether painful electrical stimulation might be considered a provocative test in producing MLCEPs and predictor of patient's outcomes after cardiac arrest. METHODS: Retrospective pilot study. SEPs with and without pain-related electrical stimulation in both median nerves were recorded in 17 patients with post anoxic coma after cardiac arrest. Glasgow Coma Scale, electroencephalograms, heart rate and blood pressure changes were also recorded at the same time. Three months after cardiac arrest the same measures with inclusion of Glasgow Outcome Scale Extended were also performed only in the remaining patients with severe neurological outcome. No one intervention was made. RESULTS: Patients who showed MLCEPs had a good outcome, while patients without N20/P25 SEPs but with increases in blood pressure remained in a vegetative state. Patients who did not show N20/P25 SEPs and increase in blood pressure died within one week. Only one patient who showed N20/P25 SEPs was minimally conscious. CONCLUSION: These preliminary data suggest that MLCEPs elicited by painful electrical stimulation seem to be a sensitive method to predict the neurological outcome of patients in the acute phase of coma. Blood pressure response might be a prognostic physiological measure of survival in the vegetative state in patients without N20/P25 SEPs.

Malignant fibrous histiocytoma of the mandible.

Malignant fibrous histiocytoma (MFH) of the mandible is rare. We describe a case of MFH of the mandible with metastatic disease to the lungs. To our knowledge only 30 cases have been reported prior to the present case. In previously reported cases, there was a strong tendency for the posterior portion of the mandible to be affected by MFH; ours is only the second reported case involving the anterior portion of the mandible. We review the diagnosis, pathology, and treatment of this rare malignancy.

NF2-deficient cells depend on the Rac1-canonical Wnt signaling pathway to promote the loss of contact inhibition of proliferation.

The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a membrane/cytoskeleton protein necessary for the maintenance of contact inhibition of growth in cells. Bi-allelic inactivation of NF2 is known to cause multiple cancers in both humans and mice. However, the mechanism through which merlin exerts its tumor-suppressive function remains obscure. In this report, we show that NF2 knockout mouse embryonic fibroblasts lost contact inhibition of cell proliferation and contained significantly increased canonical Wnt signaling. Inhibition of Rac1, the activity of which is inversely regulated by NF2, through the use of a dominant-negative mutant, small hairpin RNA or a small molecule inhibitor in NF2-deficient cells, was able to suppress elevated Wnt signals as shown by reduced activity of the T-cell factor 4 (TCF4) transcription factor. Dominant-negative TCF4 or Rac1 mutant, as well as a small molecule inhibition of Wnt, were able to curb NF2 deficiency-elicited cell proliferation at the confluent state. Thus, Rac1-mediated canonical Wnt signaling is essential for the loss of contact inhibition in NF2-deficient cells.

Rac1 GTPase: a "Rac" of all trades.

Rac1, a member of the Rho family of GTPases, is an intracellular transducer known to regulate multiple signaling pathways that control cytoskeleton organization, transcription, and cell proliferation. Deregulated expression or activation patterns of Rac1 can result in aberrant cell signaling and numerous pathological conditions. Here, we highlight the physiological functions and signaling mechanisms of Rac1 and their relevance to disease.

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function.

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) expression is regulated at both the transcriptional and post-transcriptional level in epithelial cells. The aim of this study was to characterize the effects of tyrosine phosphorylation on iNOS activity. In a human intestinal epithelial cell line stimulated with cytokines, tyrosine phosphorylation of human iNOS protein was observed after 30 min exposure to pervanadate (PV), an inhibitor of protein tyrosine phosphatases. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src tyrosine kinases, abolished the PV-induced iNOS tyrosine phosphorylation. Cotransfection of Src with iNOS cDNA in human embryonic kidney (HEK) 293 cells resulted in a threefold (P<0.001) increase of iNOS protein levels and tyrosine phosphorylation of iNOS. In the presence of Src, 76% of wild-type (wt) iNOS was redistributed to detergent-insoluble domains and iNOS activity was decreased by 28% (P<0.05) despite increased iNOS protein levels. Analysis of iNOS tyrosine mutants revealed decreased Src-induced effects in Y151F iNOS mutant. Using a GST-fusion protein containing a domain encompassing Y151, we show that Y151 is a direct substrate for active Src in vitro. These findings indicate a role for iNOS tyrosine phosphorylation in the regulation of iNOS activity and the implication of Src tyrosine kinases in this pathway.

Speech perception results in children implanted with Clarion devices: Hi-Resolution and Standard Resolution modes.

CONCLUSIONS: Formal testing showed that HiRes users seem to make significant use of acoustic information. Furthermore, from observations reported by experienced care-givers, for example, family, teachers and speech therapists, these children learn a lot from their surrounding environment. Incidental learning, which takes place when acquiring skills or knowledge through naturally occurring events, is a key become available to these deaf paediatric cochlear implant recipients. OBJECTIVE: To compare speech perception skills in children with a Clarion cochlear implant using different speech coding strategies, such as continuous interleaved sampling (CIS), simultaneous analogue stimulation (SAS) and Hi-Resolution (HiRes). MATERIAL AND METHODS: The study population comprised 40 children, 17 implanted with a Clarion Hi-Focus 1.2 and 23 with a Clarion CII. All children were pre-lingually deaf and differed in terms of age and cause of deafness. All children had undergone a trial (minimum 6 months) with hearing aids before implantation. Children implanted with a Clarion 1.2 were either CIS or SAS users [Standard Resolution mode (SRM) group]; children implanted with a Clarion CII were Hi-Resolution users [Hi-Resolution mode (HRM) group]. Findings were assessed according to-Erber's hierarchical model (detection, discrimination, identification, recognition and comprehension), making use of a battery of speech perception tests calibrated to the age of the child. Further information concerning use of the implant in everyday situations was obtained by means of the Meaningful Auditory Integration Scale (MAIS) questionnaire, which was administered to the parents. Tests were carried out prior to each fitting session, at switch-on and then at 3, 6, 9 and 12 months. Findings at pre-implantation and at 12 months follow-up were collected for both the SRM and HRM groups. Speech perception results were analysed for the SRM and HRM groups, independent of age at implantation, for five subgroups of children according to the paediatric test battery in use and for two subgroups of children, one < and one > 5 years of age. RESULTS: Clarion-implanted children using the Hi-Resolution strategy can develop better speech perception skills at 12 months post-implantation compared to children fitted with the SAS or CIS strategy. SAS or CIS users implanted before the age of 5 years tend to achieve better results at 1 year follow-up than children implanted later. In contrast, in Hi-Resolution users, a trend towards better results for recognition and comprehension tasks was observed in children implanted after 5 years of age.

Short-term study of the effect of speech coding strategy on the auditory performance of pre- and post-lingually deafened adults implanted with the Clarion CII.

OBJECTIVE: The auditory performance of cochlear implantees is linked to numerous variables, such as audiological characteristics, age and type of speech coding strategy. In recent years, many different ways of processing sounds have been developed, with possible implications for auditory performance. The purpose of this study was to evaluate the intra-individual differences of patients for speech perception tasks in quiet and in noise as a result of switching from a standard strategy [Continuous Interleaved Sampling (CIS)/Simultaneous Analog Strategy (SAS)] to Hi-Resolution (HiRes). MATERIAL AND METHODS: A total of 14 post-lingual adults implanted with the Clarion CII were selected for trials. At switch-on, six patients chose a CIS strategy and eight an SAS strategy. After an average period of 9.3 months, all patients were switched over to HiRes. All patients were tested (open-set mode) with bisyllabic words and sentences, in both quiet and noise [speech/noise ratio (SNR) = +10]. Testing was carried out after an initial period with the CIS/SAS strategy and 3 months after switching over to HiRes. RESULTS: After switch-over a significant improvement was seen for both speech in quiet (words +25.2%, sentences +10.8%) and speech in noise (words 44.8%, sentences 45.4%). CONCLUSION: Despite individual differences, all patients improved their performance with HiRes use, the greatest improvements being seen under unfavourable listening conditions (SNR = +10). Subjective improvements in speech discrimination and overall sound quality perception were reported even after the first week of HiRes use.

Cochlear implants in special cases: deafness in the presence of disabilities and/or associated problems.

The aim of the present study is to construct a reference model with the indication for the attitude, the requirements and the resources needed in order to be able to deal with deafness in the presence of disabilities or associated problems. The study group consisted of 13 adults and 18 children affected by profound deafness, with associated problems and disabilities, who were implanted with Clarion and Med-El devices. Selection criteria for candidacy to cochlear implantation and counselling, hospitalization, fitting and speech therapy/rehabilitation are described. Findings were assessed evaluating: (i) use of acoustic feedback, on the ground of Erber's model; (ii) self-sufficiency: assessed by a questionnaire; and (iii) social and family relationships: qualitative judgment based on direct observation, analysis of drawings and structured interviews with family, teachers and therapists. The whole group showed benefit from cochlear implantation, with particular satisfaction for post-lingual deaf-blind adults, as well as for subjects with associated psychopathologies and mental retardation. In conclusion, cochlear implants can improve life quality in profoundly deaf subjects with associated disabilities, increasing both listening and communication skills as well as self-sufficiency while family and social relationships tend to remain stable.

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter pylori: effect of bacterial eradication.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL-8) are positive in approximately 50% of Helicobacter pylori-related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL-8 production in H. pylori-infected asymptomatic humans, and to investigate the effect of eradication of H. pylori. MATERIALS AND METHODS: Biopsies of corpus and antrum of asymptomatic H. pylori positive and negative humans served for determination of the gastritis score and H. pylori status; iNOS was measured by reverse transcriptase polymerase chain reaction and immunohistochemistry and superoxide dismutase and catalase by immunohistochemistry. IL-8 in biopsies was assessed by enzyme-linked immunosorbent assay. RESULTS: Immunostaining of iNOS, catalase and superoxide dismutase was significantly associated with H. pylori infection and was localized to inflammatory cells. IL-8 concentrations were greater in the H. pylori positive than H. pylori negative group and decreased after bacterial eradication. A decrease in staining for iNOS and catalase was observed after H. pylori eradication. CONCLUSIONS: INOS and antioxidant enzymes are present in gastric biopsies of asymptomatic H. pylori positive humans. Eradication caused a significant decrease in staining for iNOS and catalase. These results indicate that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.