Kelly Redmond

c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP(L), but not c-FLIP(S), significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP(L) was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP(S) induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP(L)-overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

DNA Double Strand Break Repair: A Radiation Perspective

SIGNIFICANCE Ionizing radiation (IR) can induce a wide range of unique deoxyribonucleic acid (DNA) lesions due to the spatiotemporal correlation of the ionization produced. Of these, DNA double strand breaks (DSBs) play a key role. Complex mechanisms and sophisticated pathways are available within cells to restore the integrity and sequence of the damaged DNA molecules. RECENT ADVANCES Here we review the main aspects of the DNA DSB repair mechanisms with emphasis on the molecular pathways, radiation-induced lesions, and their significance for cellular processes. CRITICAL ISSUES Although the main characteristics and proteins involved in the two DNA DSB repair processes present in eukaryotic cells (homologous recombination and nonhomologous end-joining) are reasonably well established, there are still uncertainties regarding the primary sensing event and their dependency on the complexity, location, and time of the damage. Interactions and overlaps between the different pathways play a critical role in defining the repair efficiency and determining the cellular functional behavior due to unrepaired/miss-repaired DNA lesions. The repair pathways involved in repairing lesions induced by soluble factors released from directly irradiated cells may also differ from the established response mechanisms. FUTURE DIRECTIONS An improved understanding of the molecular pathways involved in sensing and repairing damaged DNA molecules and the role of DSBs is crucial for the development of novel classes of drugs to treat human diseases and to exploit characteristics of IR and alterations in tumor cells for successful radiotherapy applications.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent.

INTRODUCTION Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. METHODS The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. RESULTS RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. CONCLUSIONS These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.

An in vitro study of the radiobiological effects of flattening filter free radiotherapy treatments.

Flattening filter free (FFF) linear accelerators allow for an increase in instantaneous dose-rate of the x-ray pulses by a factor of 2-6 over the conventional flattened output. As a result, radiobiological investigations are being carried out to determine the effect of these higher dose-rates on cell response. The studies reported thus far have presented conflicting results, highlighting the need for further investigation. To determine the radiobiological impact of the increased dose-rates from FFF exposures a Varian Truebeam medical linear accelerator was used to irradiate two human cancer cell lines in vitro, DU-145 prostate and H460 non-small cell lung, with both flattened and FFF 6 MV beams. The fluence profile of the FFF beam was modified using a custom-designed Nylon compensator to produce a similar dose profile to the flattened beam (6X) at the cell surface but at a higher instantaneous dose-rate. For both cell lines there appeared to be no significant change in cell survival. Curve fitting coefficients for DU145 cells irradiated with constant average dose-rates were 6X: α = 0.09 ± 0.03, β = 0.03 ± 0.01 and 6FFF: α = 0.14 ± 0.13, β = 0.03 ± 0.02 with a significance of p = 0.75. For H460 cells irradiated with the same instantaneous dose-rate but different average dose-rate the fit coefficients were 6FFF (low dose-rate): α = 0.21 ± 0.11, 0.07 ± 0.02 and 6FFF (high dose-rate): α = 0.21 ± 0.16, 0.07 ± 0.03, with p = 0.79. The results indicate that collective damage behaviour does not occur at the instantaneous dose-rates investigated here and that the use of either modality should result in the same clinical outcome, however this will require further validation in vivo.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Conventional in vivo irradiation procedures are insufficient to accurately determine tumor responses to non-uniform radiation fields

Abstract Purpose: To determine differences in overall tumor responses measured by volumetric assessment and bioluminescence imaging (BLI) following exposure to uniform and non-uniform radiation fields in an ectopic prostate tumor model. Materials and methods: Bioluminescent human prostate tumor xenografts were established by subcutaneous implantation into male mice. Tumors were irradiated with uniform or non-uniform field configurations using conventional in vivo irradiation procedures performed using a 225 kVp generator with custom lead shielding. Tumor responses were measured using Vernier calipers and by BLI using an in vivo imaging system. Survival was defined as the time to quadroupling of pre-treatment tumor volume. Results: The correlation between BLI and tumor volume measurements was found to be different for un-irradiated (R = 0.61), uniformly irradiated (R = 0.34) and partially irradiated (R = 0.30) tumors. Uniformly irradiated tumors resulted in an average tumor growth delay of 60 days with median survival of 75 days, compared to partially irradiated tumors which showed an average growth delay of 24 days and median survival of 38 days. Conclusions: Correlation between BLI and tumor volume measurements is lower for partially irradiated tumors than those exposed to uniform dose distributions. The response of partially irradiated tumors suggests non-uniformity in response beyond physical dose distribution within the target volume. Dosimetric uncertainty associated with conventional in vivo irradiation procedures prohibits their ability to accurately determine tumor response to non-uniform radiation fields and stresses the need for image guided small animal radiation research platforms.

Investigation into the radiobiological consequences of pre-treatment verification imaging with megavoltage X-rays in radiotherapy

OBJECTIVE The aim of this study was to investigate the effect of pre-treatment verification imaging with megavoltage X-rays on cancer and normal cell survival in vitro and to compare the findings with theoretically modelled data. Since the dose received from pre-treatment imaging can be significant, the incorporation of this dose at the planning stage of treatment has been suggested. METHODS The impact of imaging dose incorporation on cell survival was investigated by clonogenic assay of irradiated DU-145 prostate cancer, H460 non-small-cell lung cancer and AGO-1522b normal tissue fibroblast cells. Clinically relevant imaging-to-treatment times of 7.5 and 15 min were chosen for this study. The theoretical magnitude of the loss of radiobiological efficacy due to sublethal damage repair was investigated using the Lea-Catcheside dose protraction factor model. RESULTS For the cell lines investigated, the experimental data showed that imaging dose incorporation had no significant impact on cell survival. These findings were in close agreement with theoretical results. CONCLUSION For the conditions investigated, the results suggest that allowance for the imaging dose at the planning stage of treatment should not adversely affect treatment efficacy. ADVANCES IN KNOWLEDGE There is a paucity of data in the literature on imaging effects in radiotherapy. This article presents a systematic study of imaging dose effects on cancer and normal cell survival, providing both theoretical and experimental evidence for clinically relevant imaging doses and imaging-to-treatment times. The data provide a firm foundation for further study into this highly relevant area of research.

Abstract A76: Characterization of CXC‐chemokine expression in colorectal cancer tissue and its role in mediating chemoresistance of colorectal cancer cells to conventional and molecularly targeted therapies

Aim of study: The aims of this work were (i) to characterize CXC‐chemokine and receptor expression in colorectal cancer (CRC) and (ii) study the role of CXC‐chemokine signalling in modulating CRC cell sensitivity to Oxaliplatin and anti‐EGFR therapy. Methods and Materials: CXC‐chemokine and receptor expression was analysed in a retrospective study of colorectal cancer biopsy specimens in stage II and stage III CRC patients (n=254) who took part in a randomized phase III study comparing adjuvant 5FU chemotherapy with surgery alone between 1994 and 1997. For the in vitro work, studies were carried out in a panel of CRC cell lines comprising HCT116 clonal derivatives and LoVo cells. CXCL8 ligand and receptor expression was assessed by flow cytometry, qRT‐PCR, western blotting and ELISA. Anti‐apoptotic and EGFR signalling was also assessed using qRT‐PCR and western blotting. NF‐κB activity was assessed by electrophoretic mobility shift assay (EMSA) and Oxaliplatin response was assessed using cell counts. Results: Expression of CXCR1, CXCR2 and CXCL1 was elevated in tumor epithelium relative to the histologically normal adjacent colorectal cells (p Conclusions: Our studies suggest CRC malignant epithelium is subject to increased autocrine CXC‐chemokine signaling compared to the adjacent normal colorectal tissue and this may reflect a role for the signaling in this disease. In‐vitro work suggests that chemotherapy‐induced CXC‐chemokine signaling may underpin CRC chemoresistance through multiple pathways. These results support a role for CXC‐chemokine signaling in the promotion of CRC and modulating sensitivity of this disease to current treatment Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A76.