Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.

Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.

Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.

Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

FUS expression alters the differentiation response to all-trans retinoic acid in NB4 and NB4R2 cells

The FUS gene is overexpressed in acute myeloid leukaemia (AML) patients and has roles in transcription and mRNA processing. We used ectopic expression of FUS and FUS antisense sequences to assess the effect of modulation of FUS expression in all-trans retinoic acid (ATRA)-sensitive (NB4) and insensitive (NB4R2) human acute promyelocytic (APL) cell lines which express the t(15:17) translocation. Growth, viability and differentiation patterns were maintained, but the expression of the FUS antisense construct in both the cell lines altered the response to ATRA: the previously ATRA-sensitive NB4 cells exhibited resistance; whilst the previously resistant NB4R2 cells showed a differentiation response to treatment.

Global changes in gene expression by the opportunistic pathogen Burkholderia cenocepacia in response to internalization by murine macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation.

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

The electrical characterization and response to hydrogen of Schottky diodes with a resistive metal electrode—rectifying an oversight in Schottky diode investigation

Schottky-barrier structures with a resistive metal electrode are examined using the 4-point probe method where the probes are connected to the metal electrode only. The observation of a significant decrease in resistance with increasing temperature (over a range of similar to 100 K) in the diode resistance-temperature (R(D)-T) characteristic is considered due to charge carrier confinement to the metal electrode at low temperature (high resistance), with the semiconductor progressively opening up as a parallel current carrying channel (low resistance) with increasing temperature due to increasing thermionic emission across the barrier. A simple model is constructed, based on thermionic emission at quasi-zero bias, that generates good fits to the experimental data. The negative differential resistance (NDR) region in the R(D)-T characteristic is a general effect and is demonstrated across a broad temperature range for a variety of Schottky structures grown on Si-, GaAs- and InP-substrates. In addition the NDR effect is harnessed in micro-scaled Pd/n-InP devices for the detection of low levels of hydrogen in an ambient atmosphere of nitrogen.

Older people's views of support services in response to elder abuse in communities across Ireland

Purpose – This paper seeks to present findings from the first all-Ireland study that consulted older people on their perceptions of interventions and services to support people experiencing abuse.

Design/methodology/approach – Utilising a grounded theory approach, 58 people aged 65 years and over took part in focus groups across Ireland. Four peer-researchers were also trained to assist in recruitment, data collection, analysis, and dissemination.

Findings – Participants identified preventative community-based approaches and peer supports as important mechanisms to support people experiencing, and being at risk of, elder abuse. Choices regarding care provision and housing, as well as opportunities for engagement in community activities where they can discuss issues with others, were identified as ways to prevent abuse.

Originality/value – The development of elder abuse services has traditionally been defined from the perspective of policy makers and professionals. This study looked at the perspective of the end-users of such services for the first time. The research also gave an active role to older people in the research process. The policy implication of the findings from this research is that enhanced attention and resources should be directed to community activities that enable older people to share their concerns informally thereby gaining confidence to seek more formal interventions when necessary.

Identification of Galanin and Its Receptor GalR1 as Novel Determinants of Resistance to Chemotherapy and Potential Biomarkers in Colorectal Cancer

Purpose: A major factor limiting the effective clinical management of colorectal cancer (CRC) is resistance to chemotherapy. Therefore, the identification of novel, therapeutically targetable mediators of resistance is vital.Experimental design: We used a CRC disease-focused microarray platform to transcriptionally profile chemotherapy-responsive and nonresponsive pretreatment metastatic CRC liver biopsies and in vitro samples, both sensitive and resistant to clinically relevant chemotherapeutic drugs (5-FU and oxaliplatin). Pathway and gene set enrichment analyses identified candidate genes within key pathways mediating drug resistance. Functional RNAi screening identified regulators of drug resistance.

Results: Mitogen-activated protein kinase signaling, focal adhesion, cell cycle, insulin signaling, and apoptosis were identified as key pathways involved in mediating drug resistance. The G-protein-coupled receptor galanin receptor 1 (GalR1) was identified as a novel regulator of drug resistance. Notably, silencing either GalR1 or its ligand galanin induced apoptosis in drug-sensitive and resistant cell lines and synergistically enhanced the effects of chemotherapy. Mechanistically, GalR1/galanin silencing resulted in downregulation of the endogenous caspase-8 inhibitor FLIP(L), resulting in induction of caspase-8-dependent apoptosis. Galanin mRNA was found to be overexpressed in colorectal tumors, and importantly, high galanin expression correlated with poor disease-free survival of patients with early-stage CRC.

Conclusion: This study shows the power of systems biology approaches to identify key pathways and genes that are functionally involved in mediating chemotherapy resistance. Moreover, we have identified a novel role for the GalR1/galanin receptor-ligand axis in chemoresistance, providing evidence to support its further evaluation as a potential therapeutic target and biomarker in CRC. Clin Cancer Res; 18(19); 5412–26. © 2012 AACR.