Risk of non-small cell lung cancer and the cytochrome P4501A1 Ile462Val polymorphism

Objective: The Ile462Val substitution in the cytochrome P450 1A1 gene (CYP1A1) results in increased enzymatic activity. Preliminary data suggesting a link between this polymorphism and lung cancer risk in Caucasians are inconsistent, reflecting small sample sizes and the relatively low frequency of the variant. Methods: The data set consisted of 1050 primary non-small cell lung cancer cases and 581 controls, a large homogenous population designed specifically to address previous inconsistencies. Patients were genotyped using a PCR-RFLP technique. Results: Carriers of the valine allele, CYP1A1*2C, (Ile/Val or Val/Val genotypes) were significantly over-represented in non-small cell lung cancer compared to controls (OR=1.9; 95% CI=1.2-2.9; p=0.005) when adjusted for confounders, particularly in women (OR=4.6; 95% CI=1.7-12.4; p=0.003). The valine variant was statistically significantly over-represented in cases of lung cancer younger than the median age (64 years) (OR=2.5; 95% CI=1.3-4.8; p=0.005) and cases with less than the median cumulative tobacco-smoke exposure (46 pack-years) (OR=2.4; 95% CI=1.3-4.7; p=0.007). Conclusions: These new data establish an association between the CYP1A1 Ile462Val polymorphism and the risk of developing non-small cell lung cancer, especially among women.

Disruption of BRCA I function results in telomere lengthening and increased anaphase bridge formation in immortalized cell lines

BRCA1 is a tumor suppressor that functions in controlling cell growth and maintaining genomic stability. BRCA1 has also been implicated in telomere maintenance through its ability to regulate the transcription of hTERT, the catalytic subunit of telomerase, resulting in telomere shortening, and to colocalize with the telomere-binding protein TRF1. The high incidence of nonreciprocal translocations in tumors arising from BRCA1 mutation carriers and Brca1-null mice also raises the possibility that BRCA1 plays a role in telomere protection. To date, however, the consequences for telomere status of disrupting BRCA1 have not been reported. To examine the role of BRCA1 in telomere regulation, we have expressed a dominant-negative mutant of BRCA1 (trBRCA1), known to disrupt multiple functions of BRCA1, in telomerase-positive mammary epithelial cells (SVCT) and telomerase-negative ALT cells (GM847). In SVCT cells, expression of trBRCA1 resulted in an increased incidence of anaphase bridges and in an increase in telomere length, but no change in telomerase activity. In GM847 cells, trBRCA1 also increased anaphase bridge formation but did not induce any change in telomere length. BRCA1 colocalized with TRF2 in telomerase-positive cells and with a small subset of ALT-associated PML bodies (APBs) in ALT cells. Together, these results raise the possibility that BRCA1 could play a role in telomere protection and suggest a potential mechanism for one of the phenotypes of BRCA1 deficient cells. (c) 2005 Wiley-Liss, Inc.

The key role of CD40 ligand in overcoming tumor-induced dendritic cell dysfunction

Overcoming dendritic cell (DC) dysfunction is a prerequisite for successful active immunotherapy against breast cancer. CD40 ligand (CD40L), a key molecule in the interface between T-lymphocytes and DCs, seems to be instrumental in achieving that goal. Commenting on our data that CD40L protects circulating DCs from apoptosis induced by breast tumor products, Lenahan and Avigan highlighted the potential of CD40L for immunotherapy. We expand on that argument by pointing to additional findings that CD40L not only rescues genuine DCs but also functionally improves populations of immature antigen-presenting cells that fill the DC compartment in patients with breast cancer.

Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8(+) T-cell function in Hodgkin lymphoma patients

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg (HRS) cells constitute only 0.5% of 10% of the diseased tissue. The surrounding cellular infiltrate is enriched with T cells that are hypothesized to modulate antitumor immunity. We show that a marker of regulatory T cells, LAG-3, is strongly expressed on infiltrating lymphocytes present in proximity to HRS cells. Circulating regulatory T cells (CD4(+) CD25(hi) CD45 ROhi, CD4(+) CTLA4(hi), and CD4(+) LAG-3(hi)) were elevated in HL patients with active disease when compared with remission. Longitudinal profiling of EBV-specific CD8(+) T-cell responses in 94 HL patients revealed a selective loss of interferon-gamma expression by CD8(+) T cells specific for latent membrane proteins 1 and 2 (LMP1/2), irrespective of EBV tissue status. Intratumoral LAG-3 expression was associated with EBV tissue positivity, whereas FOXP3 was linked with neither LAG-3 nor EBV tissue status. The level of LAG-3 and FOXP3 expression on the tumor-infiltrating lymphocytes was coincident with impairment of LMP1/2-specific T-cell function. In vitro pre-exposure of peripheral blood mono-nuclear cells to HRS cell line supernatant significantly increased the expansion of regulatory T cells and suppressed LMP-specific T-cell responses. Deletion of CD4(+) LAG-3(+) T cells enhanced LMP-specific reactivity. These findings indicate a pivotal role for regulatory T cells and LAG-3 in the suppression of EBV-specific cell-mediated immunity in HL.

Energy efficient radio access architectures for green radio:large versus small cell size deployment

In this paper new architectural approaches that improve the energy efficiency of a cellular radio access network (RAN) are investigated. The aim of the paper is to characterize both the energy consumption ratio (ECR) and the energy consumption gain (ECG) of a cellular RAN when the cell size is reduced for a given user density and service area. The paper affirms that reducing the cell size reduces the cell ECR as desired while increasing the capacity density but the overall RAN energy consumption remains unchanged. In order to trade the increase in capacity density with RAN energy consumption, without degrading the cell capacity provision, a sleep mode is introduced. In sleep mode, cells without active users are powered-off, thereby saving energy. By combining a sleep mode with a small-cell deployment architecture, the paper shows that the ECG can be increased by the factor n = (R/R) while the cell ECR continues to decrease with decreasing cell size.

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

A quantitative approach for understanding small-scale human mesenchymal stem cell culture - implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h-1) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Characterization of ALDH1A1 as a novel tumorigenic target mediating TAZ-induced lung tumorigenesis and stem cell phenotypes

Recent studies suggest that lung cancer stem cells (CSCs) may play major roles in lung cancer development, metastasis and drug resistance. Therefore, identification of lung CSC drivers may provide promising targets for lung cancer. TAZ (transcriptional co-activator with PDZ-binding motif) is a transcriptional co-activator and key downstream effector of the Hippo pathway, which plays critical roles in various biological processes. TAZ has been shown to be overexpressed in non-small cell lung cancer (NSCLC) and involved in tumorigenicity of lung epithelial cells. However, whether TAZ is a driver for lung CSCs and tumor formation in vivo is unknown. In addition, the molecular mechanism underlying TAZ-induced lung tumorigenesis remains to be determined. In this study, we provided evidence that constitutively active TAZ (TAZ-S89A) is a driver for lung tumorigenesis in vivo in mice and formation of lung CSC. Oncogenes upregulated in TAZ-overexpressing cells were identified with further validation. The most dramatically activated gene, Aldh1a1 (Aldehyde dehydrogenase 1 family member a1), a well-established CSC marker, showed that TAZ induces Aldh1a1 transcription by activating its promoter activity through interaction with the transcription factor TEA domain (TEAD) family member. Most significantly, inhibition of ALDH1A1 with its inhibitor A37 or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene knockout in lung cancer cells suppressed lung tumorigenic and CSC phenotypes in vitro, and tumor formation in mice in vivo. In conclusion, this study identified TAZ as a novel inducer of lung CSCs and the first transcriptional activator of the stem cell marker ALDH1A1. Most significantly, we identified ALDH1A1 as a critical meditator of TAZ-induced tumorigenic and CSC phenotypes in lung cancer. Our studies provided preclinical data for targeting of TAZ-TEAD-ALDH1A1 signaling to inhibit CSC-induced lung tumorigenesis and drug resistance in the future.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.

Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.

Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.

Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.