PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Calbindin 2 (CALB2) Regulates 5-Fluorouracil Sensitivity in Colorectal Cancer by Modulating the Intrinsic Apoptotic Pathway

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Delta psi(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.

PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling.

BACKGROUND:
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear.

METHODS:
To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies.

RESULTS:
The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis.

CONCLUSION:
Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.

Cotranscriptional role for Arabidopsis DICER-LIKE 4 in transcription termination.

Transcription termination is emerging as an important component of gene regulation necessary to partition the genome and minimize transcriptional interference. We have discovered a role for the Arabidopsis RNA silencing enzyme DICER-LIKE 4 (DCL4) in transcription termination of an endogenous Arabidopsis gene, FCA. DCL4 directly associates with FCA chromatin in the 3' region and promotes cleavage of the nascent transcript in a domain downstream of the canonical polyA site. In a dcl4 mutant, the resulting transcriptional read-through triggers an RNA interference–mediated gene silencing of a transgene containing the same 3' region. We conclude that DCL4 promotes transcription termination of the Arabidopsis FCA gene, reducing the amount of aberrant RNA produced from the locus.

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA(siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8. Mol Cancer Ther; 11(1); 119-31. (C) 2011 AACR.

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.

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.

EXPRESSION OF THE INFLAMMATORY REGULATOR A20 CORRELATES WITH LUNG FUNCTION IN PATIENTS WITH CYSTIC FIBROSIS

Abstract: Background: A20 and TAX1BP1 interact to negatively regulate NF-
-driven inflammation. A20 expression is altered in F508del/F508del
patients. Here we explore the effect of CFTR and CFTR genotype on A20 and
TAX1BP1expression. The relationship with lung function is also assessed.
Methods: Primary Nasal Epithelial cells (NECs) from CF patients
(F508del/F508del, n=8, R117H/F508del, n=6) and Controls (age-matched,
n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene
expression was determined by qPCR.
Results: Silencing of CFTR reduced basal A20 expression. Following LPS
stimulation A20 and TAX1BP1 expression was induced in control NECs and
reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del
had lower expression than R117H/F508del. A20, but not TAX1BP1 expression,
was proportional to FEV1 in all CF patients (r=0.968, p<0.001).
Conclusions: A20 expression is reduced in CF and is proportional to FEV1.
Pending confirmation in a larger study, A20 may prove a novel predictor
of CF inflammation/disease severity.

The phylogeny, structure and function of trematode cysteine proteases, with particular emphasis on the Fasciola hepatica cathepsin L family

Helminth parasites (nematodes, flatworms and cestodes) infect over 1 billion of the world's population causing high morbidity and mortality. The large tissue-dwelling worms express papain-like cysteine peptidases, termed cathepsins that play important roles in virulence including host entry, tissue migration and the suppression of host immune responses. Much of our knowledge of helminth cathepsins comes from studies using flatworms or trematode (fluke) parasites. The developmentally-regulated expression of these proteases correlates with the passage of parasites through host tissues and their encounters with different host macromolecules. Recent phylogenetic, biochemical and structural studies indicate that trematode cathepsins exhibit overlapping but distinct substrate specificities due to divergence within the protease active site. Here we provide an overview of the evolution, biochemistry and structure of these important enzymes and highlight how recent advances in proteomics and gene silencing techniques are allowing researchers to probe their biological functions. We focus mainly on members of the cathepsin L gene family of the animal and human pathogen, Fasciola hepatica, because of our deep understanding of their function, biochemistry and structure.

Affective disruption: Walter Benjamin and the 'history' of Ireland's industrial schools

What role do organizations play in writing history? In this paper, I address the part played by organizations in the enactment of large-scale violence, and focus on the ways in which the resulting histories come to be written. Drawing on the case of Ireland's industrial schools, I demonstrate how such accounts can act to serve the interests of those in power, effectively silencing and marginalizing weaker people. A theoretical lens that draws on ideas from Walter Benjamin and Judith Butler is helpful in understanding this; the concept of 'affective disruption' enables an exploration of how people's experiences of organizational violence can be reclaimed from the past, and protected in a continuous remembrance. Overall, this paper contributes a new perspective on the writing of organizational histories, particularly in relation to the enactment of violence. 

Explaining Prolonged Silences in Transitional Justice: The Disappeared in Cyprus and Spain

The article examines why some postconflict societies defer the recovery of those who forcibly disappeared as a result of political violence, even after a fully fledged democratic regime is consolidated. The prolonged silences in Cyprus and Spain contradict the experience of other countries such as Bosnia, Guatemala, and South Africa, where truth recovery for disappeared or missing persons was a central element of the transition to peace and democracy. Exhumations of mass graves containing the victims from the two periods of violence in Cyprus (1963–1974) and the Spanish Civil War (1936–1939) was delayed up until the early 2000s. Cyprus and Spain are well suited to explain both prolonged silences in transitional justice and the puzzling decision to become belated truth seekers. The article shows that in negotiated transitions, a subtle elite agreement links the non-instrumental use of the past with the imminent needs for political stability and nascent democratization. As time passes, selective silence becomes an entrenched feature of the political discourse and democratic institutions, acquiring a hegemonic status and prolonging the silencing of violence.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC.

A repressor of the transition to flowering in Arabidopsis is the MADS box protein FLOWERING LOCUS C (FLC). FCA, an RNA-binding protein, and FY, a homolog of the yeast RNA 3' processing factor Pfs2p, downregulate FLC expression and therefore promote flowering. FCA/FY physically interact and alter polyadenylation/3' processing to negatively autoregulate FCA. Here, we show that FCA requires FLOWERING LOCUS D (FLD), a homolog of the human lysine-specific demethylase 1 (LSD1) for FLC downregulation. FCA also partially depends on DICER-LIKE 3, involved in chromatin silencing. fca mutations increased levels of unspliced sense FLC transcript, altered processing of antisense FLC transcripts, and increased H3K4 dimethylation in the central region of FLC. These data support a close association of FCA and FLD in mediating H3K4 demethylation and thus transcriptional silencing of FLC and reveal roles for antisense RNA processing and DCL3 function in this regulation.