PDF file - 64K, unsupervised top 10 positive and negative correlated genes - SN38 inducible.

Abstract Background: BRAFV600E mutations occur in ~10% of colorectal cancer (CRC), are associated with poor survival and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT CRC. Using gene expression data from BRAFMT CRC patient samples, we recently identified that dopamine receptor degradation and unfolded protein response are dominant pathways deregulated in the BRAFMT subgroup with the poorest outcome. The aim of this study was to investigate the role of the dopamine receptor 2 (DRD2) pathway as novel target in BRAFMT CRC cells. Methods: Small molecule DRD2 antagonists ONC-201 and ONC-206 (Oncoceutics Inc) and a panel of isogenic paired and non-isogenic BRAFMT and BRAFWT cells were used. MTT, Flow Cytometry, Western blotting and caspase- 8, 3/7 activity assays were used to measure cell survival/death. DR5 cell localization was performed using flow cytometry. A compound library including small molecules approved by the FDA was used. Results: BRAFMT CRC cells were highly sensitive to the DRD2 antagonists ONC-201 and ONC-206 with IC50 values between 1.9-4.5μM and 0.16 and 0.24μM respectively. Treatment with ONC-201 and ONC-206 resulted in marked increases in expression levels of the endoplasmic reticulum stress proteins ATF4, CHOP and the active (spliced) form of XBP1 (sXBP1) (indicators of activation of the PERK and IRE1α UPR branches), and this was associated with apoptosis induction as indicated by PARP cleavage, caspase-9 cleavage and increased caspase-3/7 activity in the BRAFMT VACO432 cell line but not in the WT VT1 clone. Importantly, no significant effect on proliferation or apoptosis was obtained in the normal colon CCD-18 fibroblast cell line following treatment with ONC-201 or ONC-206. Using a small molecule compound library, we found that the taxanes paclitaxel and docetaxel resulted in strong synergy and apoptosis when combined with ONC-201 or ONC-206, in particular in BRAFMT CRC cells. Mechanistically, we found that the apoptosis induced by combined ONC-201/paclitaxel treatment was dependent on caspase-8 activation and on up-regulation of the death receptor 5 (DR5). Conclusions: Taken together, we have identified a role for DRD2 signalling in the survival of BRAFMT CRC cells. Our data support the development of DRD2 antagonists, in particular in combination with taxanes, for the treatment of BRAFMT CRC tumours. Citation Format: Arman Javadi, Nicholas Forsythe, Alaa Refaat, Jessica-Ann Weir, Hajrah Khawaja, David Waugh, Rohinton Tarapore, Joshua E. Allen, Patrick Johnston, Sandra Van Schaeybroeck. Targeting the dopamine receptor 2 in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3448.

Background: Colorectal cancer is the 3rd most common cancer in the UK, with around 40,000 new cases diagnosed annually. CRC patients have a 5-year overall survival rate of < 10% and more than 50% will die of metastatic disease. Intrinsic or acquired resistance to chemotherapeutic drugs is a major problem in CRC and developing an effective treatment strategy is therefore of the utmost importance. CRC cases harbouring RAS mutations (>50% cases) are associated with poor prognosis; this mutation has proven to be an important predictive factor for response to EGFR targeted therapies. Failure to target the RAS oncogene has resulted in a concentrated effort to discover targets within the downstream components of this pathway. In this study, we evaluate the roles of the small GTPases, RalA and RalB, as novel targets in RAS mutant (MT) CRC. The RALGDS/RAL pathway constitutes a RAS effector pathway and mediates cell survival, proliferation and tumorigenesis. RalB in particular contributes to cell survival through TBK1 signalling. Methods: We used an siRNA-based approach silencing RALA and RALB both individually and simultaneously in a panel of KRASMT and WT cells with and without the addition of the MEKi AZD6244 (Selumetinib). Knockdown efficiency and subsequent signalling events were assessed by western blotting. Flow cytometry and MTT assays were used to measure cell death and cell viability respectively. Connectivity mapping using data from microarray experiments was used to identify drugs mimicking the phenotype observed with siRALB, subsequently leading to the investigation of a TBK1 inhibitor which is currently ongoing. Results: We found that silencing RALB but not RALA, led to the greatest amount of cell death in RASMT but not WT CRC cells. In addition, a significant increase in cell death was observed when RALB silencing was combined with MEK inhibition. Cell death was found to be mediated by Caspase 8 and involved an upregulation of death receptor 5 (DR5). Furthermore, TBK1 inhibition was found to mimic the phenotype observed with siRALB. Conclusions: RalB but not RalA, is associated with cell survival and may contribute to drug resistance in RASMT CRC. Thus, the development of novel RalB-specific therapies may lead to new treatment strategies for RAS MT CRC. Legal entity responsible for the study: Queen's University Belfast Funding: Queen's University Belfast, Cancer Research UK Disclosure: All authors have declared no conflicts of interest.

Folates have been used with cytotoxic agents for decades and today they are used in hundreds of thousands of patients annually. Folate metabolism is complex. In the treatment of cancer with 5-fluorouracil, the administration of folates mechanistically leads to the formation of [6R]-5,10-methylene-tetrahydrofolate, and the increased concentration of this molecule leads to stabilization of the ternary complex comprising thymidylate synthase, 2'-deoxy-uridine-5'-monophosphate, and [6R]-5,10-methylene-tetrahydrofolate. The latter is the only natural folate that can bind directly in the ternary complex, with other folates requiring metabolic activation. Modulation of thymidylate synthase activity became central in the study of folate/cytotoxic combinations and, despite wide use, research into the folate component was neglected, leaving important questions unanswered. This article revisits the mechanisms of action of folates and evaluates commercially available folate derivatives in the light of current research. Better genomic insight and availability of new analytical techniques and stable folate compounds may open new avenues of research and therapy, ultimately bringing increased clinical benefit to patients.

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD’s α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

A catchment-scale salmon, Salmo salar L., habitat enhancement scheme was developed for the River Main (Northern Ireland) with reference to baseline habitat and electric fishing surveys. In total, 19 separate sites were enhanced using flow deflectors or random boulder addition. Habitat suitability for juvenile salmon increased at enhanced sites following the scheme, and significant changes in underlying physical habitat characteristics (particle size, depth and flow) were detected after the installation of flow deflectors. The overall index of salmon fry recruitment, monitored across the catchment, showed no change between pre- and post-enhancement periods. The mean biomass of salmon evident at individual enhancement sites (5.1 g m(-2)) was significantly higher than the mean biomass at control sites (1.2 g m(-2)). Increased densities of >0+ juvenile salmon were associated with enhanced sites relative to controls. Marking studies indicated the potential for long-range dispersal of juvenile salmon between 0+ (summer) and 1+ (summer) age classes. The potential of the scheme to enhance the local salmon stock was discussed.

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

BackgroundMutations in BRAF V600E oncogene (BRAFMT) occurs in 8-15% of colorectal cancer (CRC) patients1.This mutation constitutively activates MAPK signalling, resulting in a proliferative and survival advantage for the tumour cells and oncogenic BRAF status has been linked with poor prognosis2.Despite introduction of the BRAFMT specific inhibitor Vemurafenib in metastatic melanoma3, there is no effective treatment strategy for BRAFMT CRC patients.This study aimed to assess the effectiveness of Ganetespib (HSP90 inhibitor), the multi-kinase inhibitor (CRAF/ VEGFR/PDGFR) Sorafenib and the BRAFMT inhibitor Vemurafenib in BRAFMT CRC cell line models. MethodsBRAF MT RKO F6-8 (MT/WT) and isogenic wild-type T29 (null/WT) cell lines were used.MTT assays were used to determine IC50 values.Protein expression was determined by Western Blotting.Levels of apoptotic cells were assessed by flow cytometry.

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1–3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors’.