Circulating microRNAs are associated with docetaxel chemotherapy outcome in castration-resistant prostate cancer.

BACKGROUND: Docetaxel is the first-line chemotherapy for castration-resistant prostate cancer (CRPC). However, response rates are ∼50% and determined quite late in the treatment schedule, thus non-responders are subjected to unnecessary toxicity. The potential of circulating microRNAs as early biomarkers of docetaxel response in CRPC patients was investigated in this study. METHODS: Global microRNA profiling was performed on docetaxel-resistant and sensitive cell lines to identify candidate circulating microRNA biomarkers. Custom Taqman Array MicroRNA cards were used to measure the levels of 46 candidate microRNAs in plasma/serum samples, collected before and after docetaxel treatment, from 97 CRPC patients. RESULTS: Fourteen microRNAs were associated with serum prostate-specific antigen (PSA) response or overall survival, according to Mann-Whitney U or log-rank tests. Non-responders to docetaxel and patients with shorter survival generally had high pre-docetaxel levels of miR-200 family members or decreased/unchanged post-docetaxel levels of miR-17 family members. Multivariate Cox regression with bootstrapping validation showed that pre-docetaxel miR-200b levels, post-docetaxel change in miR-20a levels, pre-docetaxel haemoglobin levels and visceral metastasis were independent predictors of overall survival when modelled together. CONCLUSIONS: Our study suggests that circulating microRNAs are potential early predictors of docetaxel chemotherapy outcome, and warrant further investigation in clinical trials.

c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer.

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

Cyclin D1 overexpression induces progestin resistance in T-47D breast cancer cells despite p27(Kip1) association with cyclin E-Cdk2.

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.

The effect of ribavirin to treat previously healthy infants admitted with acute bronchiolitis on acute and chronic respiratory morbidity.

The role of ribavirin in the treatment of acute bronchiolitis is controversial. It has been suggested that the use of ribavirin may be of benefit during the acute illness and may reduce subsequent recurrent respiratory morbidity. This randomized, double-blind, placebo-controlled study was designed to determine whether ribavirin administered during the acute illness would have an influence on respiratory morbidity during both the acute illness and during the following year. Bronchial reactivity 6 months after the acute illness was also assessed. Forty previously well infants with moderately severe acute bronchiolitis were recruited during three winter epidemics. Subjects received study medication for 18 h a day. Management was otherwise unaltered. Subjects were evaluated daily by the investigator and subsequently assessed at 6 weeks, 6 months and 1 year following the acute illness. Assessment of bronchial hyper-responsiveness was assessed at 6 months of age using total body plethysmography and an established ultra-sonically nebulized distilled water challenge. A total of 40 patients (21 ribavirin, 19 placebo) were entered into the study. The two groups did not differ with respect to age, gender or clinical severity on entry to the trial. No significant differences were identified in the rate of clinical improvement over the first 24 h, the time to discharge, bronchial responsiveness at 6 months of age, frequency of significant respiratory symptoms over the first year of life and the frequency of prescribed bronchodilators and inhaled steroids during the year of follow-up. This study was unable to demonstrate any clinical benefit from the use of ribavirin in the acute illness or during subsequent follow-up for 1 year.

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells.

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.

Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells.

The G1 cyclins, cyclin D1 and E, are rate limiting for progression through G1 phase of the cell cycle in breast epithelial cells and are oncogenic when expressed in the mammary epithelium of transgenic mice. These genes are frequently overexpressed in clinical breast cancer where overexpression appears to be associated with specific disease phenotypes, altered responsiveness to therapeutic intervention and patient survival. In order to investigate the functional correlates of cyclin D1 and cyclin E overexpression we employed a panel of normal, immortalized and neoplastic breast epithelial cell lines to examine the relationships between cyclin gene expression, cyclin-CDK complex formation and CDK activity. In agreement with earlier studies cyclin D1 and E expression varied over an approximately tenfold range among the 18 cell lines studied. There was no apparent relationship, however, between cyclin D1 expression and the in vitro activity of its major kinase partner, Cdk4, although MDA-MB-134 cells displayed the highest level of both cyclin D1 expression and Cdk4 activity. Similarly, there was no significant relationship between cyclin E expression and cyclin E-Cdk2 activity. Fractionation of whole cell lysates by gel filtration chromatography revealed that approximately 90% of the cyclin E protein was present in inactive complexes containing the CDK inhibitors p21 and p27. Much of the small fraction of active cyclin E protein was of very high apparent molecular mass, >400 kDa, suggesting that formation of these complexes is a more important determinant of cyclin E-Cdk2 activity than cyclin E abundance. These data suggest that properties of cyclins D1 and E in addition to their ability to activate Cdk4 and Cdk2 may contribute to the effects of overexpression on the breast cancer phenotype.

Mechanisms of cyclin-dependent kinase inactivation by progestins.

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of approximately 120 and approximately 200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.

Respiratory status and allergy nine to 10 years after acute bronchiolitis.

In order to evaluate further the relationship between acute bronchiolitis in infancy and subsequent respiratory problems, children prospectively followed up from the time of their admission to hospital were reviewed along with a group of matched controls recruited at the previous five and a half year assessment. Sixty one index children and 47 controls took part. The groups were well matched for age, height, parental smoking, and social class. Although the prevalence of respiratory symptoms had fallen when related to the previous review, there remained an excess of coughing (48 and 17% in index and control children respectively; odds ratio 4.02) and wheezing (34 and 13% in index and control children respectively; odds ratio 3.59). Bronchodilator therapy was used by 33% of index children compared with 3% of controls. Lung function tests revealed no significant differences in the measurements of lung growth-for example, forced vital capacity, functional residual capacity, and total lung capacity-but the index children had significant reductions in measurements of airways obstruction-for example, forced expiratory volume in one second, maximum expiratory flow at 75, 50 and 25% of vital capacity, and airways resistance. Family history and personal skin tests showed no excess of atopy in the index group. This study supports the claim that the excess respiratory symptoms after acute bronchiolitis are not due to familial or personal susceptibility to atopy.

Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21.

Progestin antagonists inhibit the proliferation of progesterone receptor-positive cells, including breast cancer cells, by G1 phase-specific actions, but the molecular targets involved are not defined. Reduced phosphorylation of pRB, a substrate for G1 cyclin-dependent kinases (CDKs) in vivo, was apparent after 9 h treatment of T-47D breast cancer cells with the antiprogestins RU 486 or ORG 31710, accompanying changes in S phase fraction. Although the abundance of cyclin D1, Cdk4, and Cdk6 did not decrease cyclin D1-associated kinase activity was reduced by approximately 50% at 9-18 h. Similarly, cyclin E-associated kinase activity decreased by approximately 60% at 12-24 h in the absence of significant changes in the abundance of cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein abundance and was present at increased levels in cyclin D1 and cyclin E complexes at times when their kinase activity was decreased. Increased p21 protein abundance was observed in another antiprogestin-sensitive cell line, BT 474, but not in two breast cancer cell lines insensitive to antiprogestins. These data suggest increased p21 abundance and concurrent inhibition of CDK activity as a mechanism for antiprogestin induction of growth arrest. Antiprogestin effects on proliferation were markedly reduced after ectopic expression of cyclin D1, indicating that inhibition of cyclin D1 function is a critical element in antiprogestin inhibition of proliferation. However, these data also implicate regulation of cyclin E function in antiprogestin regulation of cell cycle progression.

Nucleotide variation in the cytidine triphosphate synthetase gene of Giardia duodenalis.

The cytidine triphosphate synthetase genes from three diverse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including divergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most divergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.

Tryptase and IgE concentrations in the respiratory tract of infants with acute bronchiolitis.

It has been proposed that a specific IgE response contributes to the immunopathology of acute respiratory syncytial virus (RSV) bronchiolitis but previous work has been difficult to replicate. Indirect evidence that might support this contention was sought by measuring total IgE concentrations in bronchoalveolar lavage (BAL) samples obtained from intubated infants and by attempting to detect mRNA for IgE in cells obtained from both the upper and lower respiratory tract. Evidence of significant mast cell activation was sought by measuring tryptase concentrations in BAL fluid and serum. Detectable concentrations of IgE were found in two of seven BAL samples obtained more than five days after intubation and mRNA for IgE was demonstrated in three of six BAL samples and three of six samples obtained from the upper respiratory tract. Tryptase was detectable in 11 of 12 BAL samples with the two highest values detected on day 1. These values were raised compared with control samples but were not such to suggest that mast cell degranulation is the major contributor to the inflammatory process. These results suggest that IgE may be produced in the airways of infants in response to RSV infection. The relationships between IgE production, RSV infection, and symptoms of acute bronchiolitis remain obscure.

Analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection.

To study the cellular infiltrate that occurs within the airways of infants with respiratory syncytial virus bronchiolitis, samples of airways secretions were obtained by bronchial lavage from the lower respiratory tract of infants ventilated for this condition and from the upper airway of non-intubated infants with this disorder using nasopharyngeal aspirates. Cytospin samples were prepared so that differential cell counts could be performed on the cells obtained and alkaline phosphatase-antialkaline phosphatase immunocytochemical analysis of lymphocyte subsets was carried out using a panel of monoclonal antibodies, which included anti-CD3, anti-CD4, anti-CD8, anti-CD19, and anti-TcR gamma delta. Results from the lower and upper airways were similar. Large numbers of inflammatory cells were obtained, of which neutrophils accounted for a median of 93% in the upper airway and 76% in the lower airway. The numbers of CD8 positive cells detected were small and consistently less than CD4 positive cells, median CD4:CD8 ratios being 22.5:1 and 15:1 for the lower and upper airways. CD19 positive cells were rarely observed and no gamma delta positive lymphocytes were detected. These results indicate that neutrophils probably play a major part in causing symptoms in these infants. They do not support the concept that excessive lymphocyte mediated cytotoxic activity is principally responsible for the pathology in respiratory syncytial virus bronchiolitis.

An assessment of a new breath actuated inhaler device in acutely wheezy children.

A randomised double blind, two period cross over study was designed to compare the ability of 51 hospitalised asthmatics with acute exacerbations to use each of two inhalers. The inhalers compared were a new breath actuated metered dose inhaler, the Autohaler inhalation device, and a dry powder device, the Rotahaler. Preassessment data included the measurement of peak inspiratory flow rate (PIFR), peak expiratory flow rate (PEFR), pulse rate, and oxygen saturation. Therapeutic response to each inhaler was compared by measurement of PEFR, oxygen saturation, and pulse rate. PIFR was sufficient in all children to fire the Autohaler, including the youngest. No significant difference was found between the two inhalers as assessed by PEFR. However the Autohaler inhalation device could be actuated 99/100 times successfully compared with 74 for the Rotahaler. There was a consistent, but clinically insignificant, increase in pulse rate after use of the Rotahaler compared with the Autohaler. All 11 patients under 6 years of age failed to empty the Rotahaler but five of these patients received a significant benefit from using the Autohaler compared with after the Rotahaler. A significant drop in oxygen saturation was observed 15 minutes after use of either inhaler. This may at times reach levels of clinical importance.

Respiratory status and allergy after bronchiolitis.

As part of a long term prospective study, 73 children who had been admitted to hospital with viral bronchiolitis as infants, were reviewed 5.5 years later and compared with a carefully matched control group. In the postbronchiolitis group, there was a highly significant increase in respiratory symptoms including wheezing (42.5% v 15.0%, relative risk = 2.8). Although atopy in the family was not significantly increased in the index group, personal atopy was more prevalent. However, personal atopy was not significantly more prevalent in the symptomatic postbronchiolitis, compared with those who were symptom free, and so did not account for the high prevalence of postbronchiolitis wheezing in this cohort. In addition, in a stepwise logistic regressional model, bronchiolitis remained a significant predictor of wheezing after adjusting for potential confounding variables, including atopy. Bronchial responsiveness to histamine was significantly increased in the index group. However, no significant relationship of positive tests to wheezing could be demonstrated, and a high rate of positive responses was noted in the controls.

Nebulised water as a bronchoconstricting challenge in infancy.

The pulmonary response to inhalation challenge with nebulised distilled water was measured in 100 sedated infants with a history of wheeze. Lung function was measured by total body plethysmography. Satisfactory results were obtained in 88 infants. Fifty one were considered to have responded by developing a greater than 20% decrease in specific conductance (sGaw) after nebulised water. Thirty two of these infants had previously been challenged with nebulised saline before but only one showed a greater than 20% decrease in sGaw after saline. Twenty infants who developed signs of bronchoconstriction after challenge with nebulised water were rechallenged 20 minutes later. After the initial challenge a fall in sGaw of greater than 20% was found in 19 of the 20. After a second challenge with nebulised water only 15 (75%) showed a 20% or greater decrease in sGaw. Nine of the 20 infants remained sedated and were rechallenged for a third time. Eight showed a greater than 20% decrease in sGaw. This study indicates that approximately 60% of infants with a history of wheeze will bronchoconstrict in response to inhaled nebulised water and that up to 75% show no evidence of a subsequent refractory period to inhaled water challenge.

Nebulised sodium cromoglycate in infancy: airway protection after deterioration.

Over a two year period 100 infants with histories of wheeze were challenged with nebulised water. They were sedated and lung function measured by total body plethysmography. Thirteen of the 53 infants who developed bronchoconstriction after challenge with nebulised water were given nebulised sodium cromoglycate and rechallenged with nebulised water. All infants were initially challenged with normal saline, after which there was no significant change in lung function. After challenge with nebulised water and sodium cromoglycate there were significant decreases in specific conductance compared with those found after challenge with normal saline. After rechallenge with nebulised water there was no deterioration in lung function. Although sodium cromoglycate caused a deterioration in lung function in these infants, it protected their airways from challenge with nebulised water.

Interferon alfa, infectious virus, and virus antigen secretion in respiratory syncytial virus infections of graded severity.

Interferon alfa was measured by an immunoradiometric assay in the nasopharyngeal secretions of a group of infants admitted to hospital with respiratory syncytial virus infection. Virus replication in the upper respiratory tract was assessed by infectivity assay and by an enzyme linked immunoadsorbent assay for the viral fusion protein on the same nasopharyngeal secretions. All infants were examined daily while in hospital and allocated a score based upon a subjective assessment of the severity of their illness. There was no significant correlation between interferon, virus, or fusion (F) protein secretion and severity of illness or age of infant. It is concluded that poor interferon alfa secretion does not underly the susceptibility of infants to severe infections with this virus.

IgG and IgM antibodies to viral glycoproteins in respiratory syncytial virus infections of graded severity.

Serum antibodies to the fusion (F) and large glycoprotein (G) of respiratory syncytial virus in the serum of 57 infected infants were measured by enzyme linked immunosorbent assay (ELISA). Most serum samples taken at the time of admission to hospital contained antibodies to both glycoproteins, and overall there was no significant evidence of a selective deficiency of antibody to either viral antigen. Less than a quarter of the infants showed rising IgG antibody titres to either glycoprotein after infection, whereas over threequarters produced an IgM response. There was a significant correlation between IgG response to viral glycoproteins and the age of the infant. The correlation of age with the IgM response was less pronounced, and there was no correlation between serum IgG antibody derived transplancentally in the acute phase of infection and IgM response to either glycoprotein. Neither IgG or IgM responses correlated with a clinical assessment of the severity of infection in the infants. IgM responses, however, were weakly correlated with reduced secretion of infectious virus in the upper respiratory tract.