Apoptosis-Inducing TNF Superfamily Ligands for Cancer Therapy

Cancer is a complex disease with apoptosis evasion as one of its hallmarks; therefore, apoptosis induction in transformed cells seems a promising approach as a cancer treatment. TNF apoptosis-inducing ligands, which are naturally present in the body and possess tumoricidal activity, are attractive candidates. The most studied proteins are TNF-α, FasL, and TNF-related apoptosis-inducing ligand (TRAIL). Over the years, different recombinant TNF family-derived apoptosis-inducing ligands and agonists have been designed. Their stability, specificity, and half-life have been improved because most of the TNF ligands have the disadvantages of having a short half-life and affinity to more than one receptor. Here, we review the outlook on apoptosis-inducing ligands as cancer treatments in diverse preclinical and clinical stages and summarize strategies of overcoming their natural limitations to improve their effectiveness

Modulating CRISPR/Cas9 genome-editing activity by small molecules

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genome engineering has become a standard procedure for creating genetic and epigenetic changes of DNA molecules in basic biology, biotechnology, and medicine. However, its versatile applications have been hampered by its overall low precise gene modification efficiency and uncontrollable prolonged Cas9 activity. Therefore, overcoming these problems could broaden the therapeutic use of CRISPR/Cas9-based technologies. Here, we review small molecules with the clinical potential to precisely modulate CRISPR/Cas9-mediated genome-editing activity and discuss their mechanisms of action. Based on these data, we suggest that direct-acting small molecules for Cas9 are more suitable for precisely regulating Cas9 activity. These findings provide useful information for the identification of novel small-molecule enhancers and inhibitors of Cas9 and Cas9-associated endonucleases

CRISPR/Cas9 for overcoming drug resistance in solid tumors

Objectives: In this review, we focus on the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome editing system, in the identification of resistance mechanisms and in overcoming drug resistance in the most frequent solid tumors. Data acquisition: Data were collected by conducting systematic searching of scientific English literature using specific keywords such as “cancer”, “CRISPR” and related combinations. Results: The review findings revealed the importance of CRISPR/Cas9 system in understanding drug resistance mechanisms and identification of resistance-related genes such as PBRM1, SLFN11 and ATPE1 in different cancers. We also provided an overview of genes, including RSF1, CDK5, and SGOL1, whose disruption can synergize with the currently available drugs such as paclitaxel and sorafenib. Conclusion: The data suggest CRISPR/Cas9 system as a useful tool in elucidating the molecular basis of drug resistance and improving clinical outcomes. [Figure not available: see fulltext.

CRISPR/Cas9:A powerful tool for identification of new targets for cancer treatment

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest here that CRISPR/Cas9 is an effective system to distinguish between mutant and wild-type alleles in cancer. We show that several new potential therapeutic targets, such as CD38, CXCR2, MASTL, and RBX2, as well as several noncoding (nc)RNAs have been identified using CRISPR/Cas9 technology. We also discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic

CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation

Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR(-/-) cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma

In vivo evaluation of [F-18]FEAnGA-Me:a PET tracer for imaging beta-glucuronidase (beta-GUS) activity in a tumor/inflammation rodent model

Introduction: The PET tracer, 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-beta-D-glucopyronuronate ([F-18]FEAnGA), was recently developed for PET imaging of extracellularl beta-glucuronidase (beta-GUS). However,[F-18]FEAnGA exhibited rapid renal clearance, which resulted in a relatively low tracer uptake in the tumor. To improve the pharmacokinetics of [F-18]FEAnGA, we developed its more lipophilic methyl ester analog, [F-18]FEAnGA-Me. Methods: [F-18]FEAnGA-Me was obtained by alkylation of the O-protected glucuronide methyl ester precursor with [F-18]-fluoroethylamine ([F-18]FEA), followed by removal of the acetate protecting groups with NaOMe/MeOH. The PET tracer was evaluated by in vitro and in vivo studies. Results: [F-18]FEAnGA-Me was obtained in 5%-10% overall radiochemical yield. It is 10-fold less hydrophilic than [F-18]FEAnGA and it is stable in PBS and in the presence of beta-GUS for 1 h. However, in the presence of esterase or plasma [F-18]FEAnGA-Me is converted to [F-18]FEAnGA, and subsequently converted to [F-18]FEA by beta-GUS. MicroPET studies in Wistar rats bearing a C6 glioma and a sterile inflammation showed similar uptake in tumors after injection of either [F-18]FEAnGA-Me or [F-18]FEAnGA. Both tracers had a rapid two-phase clearance of total plasma radioactivity with a half-life of 1 and 8 min. The [F-18]FEAnGA fraction generated from [F-18]FEAnGA-Me by in vivo hydrolysis had a circulation half-life of 1 and 11 min in plasma. Similar distribution volume in the viable part of the tumor was found after injection of either [F-18]FEAnGA-Me or [F-18]FEAnGA. Conclusion: The imaging properties of [F-18]FEAnGA-Me were not significantly better than those of [F-18]FEAnGA. Therefore, other strategies should be applied in order to improve the kinetics of these tracers. (C) 2012 Elsevier Inc. All rights reserved

D-dopachrome tautomerase contributes to lung epithelial repair via atypical chemokine receptor 3-dependent Akt signaling

BACKGROUND: Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD. METHODS: We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation. FINDINGS: We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells. INTERPRETATION: In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

AbstractThe increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases