Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PURPOSE: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown. PATIENTS AND METHODS: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively). RESULTS: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031). CONCLUSION: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

DED or alive: assembly and regulation of the death effector domain complexes.

Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.

Nanoencapsulation of ABT-737 and camptothecin enhances their clinical potential through synergistic antitumor effects and reduction of systemic toxicity.

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.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.

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.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer.

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.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer.

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.

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma.

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.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer.

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

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'.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing.

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Anti-apoptotic mechanisms of drug resistance in cancer.

Resistance to chemotherapy is a major obstacle in the treatment of cancer. Despite the advent of new chemotherapies and molecular-targeted therapies, approximately 90% of patients with metastatic cancer succumb to their disease. Drug resistance, either acquired or intrinsic, often prevents tumour cells from undergoing sufficient levels of programmed cell death or apoptosis, resulting in cancer cell survival and treatment failure. In pre-clinical disease models, agents that target the apoptotic pathway have been shown to sensitize tumour cells to chemotherapy and radiotherapy. Such therapies include small molecule inhibitors and antisense strategies that inhibit the activity of anti-apoptotic proteins, or treatment with recombinant pro-apoptotic proteins or antibodies that can activate the apoptotic pathway. This review will discuss apoptosis and the mechanisms by which it can become dysregulated in human cancer. In addition, novel therapeutic strategies that target key components of the apoptotic machinery will be discussed.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells.

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.

c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIP(L) and c-FLIP(S) with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53(+/+), RKO), null (HCT116p53(-/-)) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIP(L), but not c-FLIP(S), potently inhibited apoptosis induced by chemotherapy in HCT116p53(+/+) cells, suggesting that c-FLIP(L) was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIP(L) synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAIL-independent, DR5- and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

Molecular mechanisms of drug resistance.

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Tumours may be intrinsically drug-resistant or develop resistance to chemotherapy during treatment. Acquired resistance is a particular problem, as tumours not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different mechanisms of action. Resistance to chemotherapy is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumour cells may also reduce the effectiveness of chemotherapy in the adjuvant setting. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanisms of drug resistance that operate to reduce drug sensitivity in cancer cells. Drug resistance can occur at many levels, including increased drug efflux, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis. Advances in DNA microarray and proteomic technology, and the ongoing development of new targeted therapies have opened up new opportunities to combat drug resistance. We are now able to characterize the signalling pathways involved in regulating tumour cell response to chemotherapy more completely than ever before. This will facilitate the future development of rational combined chemotherapy regimens, in which the newer targeted therapies are used in combination with cytotoxic drugs to enhance chemotherapy activity. The ability to predict response to chemotherapy and to modulate this response with targeted therapies will permit selection of the best treatment for individual patients.

Nuclear thymidylate synthase expression, p53 expression and 5FU response in colorectal carcinoma.

Thymidylate synthase (TS) is a key enzyme in DNA synthesis and is inhibited by metabolites of the chemotherapeutic agent 5-fluorouracil (5FU). Nuclear expression of TS in human tissue in vivo has not been characterised and its clinicopathological correlates in malignancy are unknown. 52 cases of primary colorectal carcinoma (CRC) and 24 cases of matched metastatic carcinoma were studied immunohistochemically using the monoclonal antibody TS106. The degree of nuclear TS immunostaining correlated closely with levels of TS mRNA expression amongst 10 CRCs studied. Strong nuclear immunostaining was seen in normal basal crypt colonocytes and germinal centre cells, and in a varying proportion of adenocarcinoma cells. Amongst the primary carcinomas, higher TS nuclear expression was associated with prominent extracellular mucin production and right-sided location. Higher TS nuclear expression also showed a significant association with poorer response to protracted venous infusional 5FU therapy. There was no clear association between TS nuclear expression and Ki67 or p53 expression assessed immunohistochemically. There was a strong positive correlation between TS nuclear expression in primary and metastatic CRC but the latter generally showed higher expression than matched primary tumour tissue. These findings confirm the nuclear expression of TS protein in human cells in vivo and provide new insight into how such expression may relate to the behaviour of CRCs.

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies.

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.