Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy.

BACKGROUND: Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood. METHODS: The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer. RESULTS: In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response. CONCLUSION: HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors.

Protein kinase D drives the secretion of invasion mediators in triple-negative breast cancer cell lines.

The protein kinase D (PKD) family members regulate the fission of cargo vesicles at the Golgi complex and play a pro-oncogenic role in triple-negative breast cancer (TNBC). Whether PKD facilitates the secretion of tumor-promoting factors in TNBC, however, is still unknown. Using the pharmacological inhibition of PKD activity and siRNA-mediated depletion of PKD2 and PKD3, we identified the PKD-dependent secretome of the TNBC cell lines MDA-MB-231 and MDA-MB-468. Mass spectrometry-based proteomics and antibody-based assays revealed a significant downregulation of extracellular matrix related proteins and pro-invasive factors such as LIF, MMP-1, MMP-13, IL-11, M-CSF and GM-CSF in PKD-perturbed cells. Notably, secretion of these proteins in MDA-MB-231 cells was predominantly controlled by PKD2 and enhanced spheroid invasion. Consistently, PKD-dependent secretion of pro-invasive factors was more pronounced in metastatic TNBC cell lines. Our study thus uncovers a novel role of PKD2 in releasing a pro-invasive secretome.

Secretome processing for proteomics: A methods comparison.

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r = 0.94, CV = 19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS.

A Robust and Clinically Applicable Sample Preparation Protocol for Urinary Extracellular Vesicle Isolation Suitable for Mass Spectrometry-Based Proteomics.

Urinary extracellular vesicles (uEVs) are a rich source of noninvasive protein biomarkers. However, for translation to clinical applications, an easy-to-use uEV isolation protocol is needed that is compatible with proteomics. Here, we provide a detailed description of a quick and clinical applicable uEV isolation protocol. We focus on the isolation procedure and subsequent in-depth proteome characterization using LC-MS/MS-based proteomics. As an example, we show how differential analyses can be performed using urine samples obtained from prostate cancer patients, compared to urine from controls.

Integration of stool microbiota, proteome and amino acid profiles to discriminate patients with adenomas and colorectal cancer.

BACKGROUND: Screening for colorectal cancer (CRC) reduces its mortality but has limited sensitivity and specificity. Aims We aimed to explore potential biomarker panels for CRC and adenoma detection and to gain insight into the interaction between gut microbiota and human metabolism in the presence of these lesions. METHODS: This multicenter case-control cohort was performed between February 2016 and November 2019. Consecutive patients ≥18 years with a scheduled colonoscopy were asked to participate and divided into three age, gender, body-mass index and smoking status-matched subgroups: CRC (n = 12), adenomas (n = 21) and controls (n = 20). Participants collected fecal samples prior to bowel preparation on which proteome (LC-MS/MS), microbiota (16S rRNA profiling) and amino acid (HPLC) composition were assessed. Best predictive markers were combined to create diagnostic biomarker panels. Pearson correlation-based analysis on selected markers was performed to create networks of all platforms. RESULTS: Combining omics platforms provided new panels which outperformed hemoglobin in this cohort, currently used for screening (AUC 0.98, 0.95 and 0.87 for CRC vs controls, adenoma vs controls and CRC vs adenoma, respectively). Integration of data sets revealed markers associated with increased blood excretion, stress- and inflammatory responses and pointed toward downregulation of epithelial integrity. CONCLUSIONS: Integrating fecal microbiota, proteome and amino acids platforms provides for new biomarker panels that may improve noninvasive screening for adenomas and CRC, and may subsequently lead to lower incidence and mortality of colon cancer.

Secreted protein markers in oral squamous cell carcinoma (OSCC).

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a main cause of oral cancer mortality and morbidity in central south Asia. To improve the clinical outcome of OSCC patients, detection markers are needed, which are preferably non-invasive and thus independent of a tissue biopsy. METHODS: In the present study, we aimed to identify robust candidate protein biomarkers for non-invasive OSCC diagnosis. To this end, we measured the global protein profiles of OSCC tissue lysates to matched normal adjacent mucosa samples (n = 14) and the secretomes of nine HNSCC cell lines using LC-MS/MS-based proteomics. RESULTS: A total of 5123 tissue proteins were identified, of which 205 were robustly up- regulated (p-value < 0.01, fold change > + 2) in OSCC-tissues compared to normal adjacent tissues. The biological process "Secretion" was highly enriched in this set of proteins. Other upregulated biological pathways included "Unfolded Protein Response", "Spliceosomal complex assembly", "Protein localization to endosome" and "Interferon Gamma Response". Transcription factor analysis implicated Creb3L1, ESRRA, YY, ELF2, STAT1 and XBP as potential regulators. Of the 205 upregulated tissue proteins, 132 were identified in the cancer cell line secretomes, underscoring their potential use as non-invasive biofluid markers. To further prioritize our candidate markers for non-invasive OSCC detection, we integrated our data with public biofluid datasets including OSCC saliva, yielding 25 candidate markers for further study. CONCLUSIONS: We identified several key proteins and processes that are associated with OSCC tissues, underscoring the importance of altered secretion. Cancer-associated OSCC secretome proteins present in saliva have potential to be used as novel non-invasive biomarkers for the diagnosis of OSCC.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Lipopolysaccharide-regulated secretion of soluble and vesicle-based proteins from a panel of colorectal cancer cell lines.

PURPOSE: To mimic the perioperative microenvironment where bacterial products get in contact with colorectal cancer (CRC) cells and study its impact on protein release, we exposed six CRC cell lines to lipopolysaccharide (LPS) and investigated the effect on the secretome using in-depth mass spectrometry-based proteomics. EXPERIMENTAL DESIGN: Cancer cell secretome was harvested in bio-duplicate after LPS treatment, and separated in EV and soluble secretome (SS) fractions. Gel-fractionated proteins were analysed by label-free nano-liquid chromatography coupled to tandem mass spectrometry. NF-κB activation, triggered upon LPS treatment, was evaluated. RESULTS: We report a CRC secretome dataset of 5601 proteins. Comparison of all LPS-treated cells with controls revealed 37 proteins with altered abundance in the SS, including RPS25; and 13 in EVs, including HMGB1. Comparing controls and LPS-treated samples per cell line, revealed 564 significant differential proteins with fold-change >3. The LPS-induced release of RPS25 was validated by western blot. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial endotoxin has minor impact on the global CRC cell line secretome, yet it may alter protein release in a cell line-specific manner. This modulation might play a role in orchestrating the development of a permissive environment for CRC liver metastasis, especially through EV-communication.

Feasibility of phosphoproteomics to uncover oncogenic signalling in secreted extracellular vesicles using glioblastoma-EGFRVIII cells as a model.

Cancer cells secrete extracellular vesicles (EVs) that contain molecular information, including proteins and RNA. Oncogenic signalling can be transferred via the cargo of EVs to recipient cells and may influence the behaviour of neighbouring cells or cells at a distance. This cargo may contain cancer drivers, such as EGFR, and also phosphorylated (activated) components of oncogenic signalling cascades. Till date, the cancer EV phosphoproteome has not been studied in great detail. In the present study, we used U87 and U87EGFRvIII cells as a model to explore EV oncogenic signalling components in comparison to the cellular profile. EVs were isolated using the VN96 ME-kit and subjected to LC-MS/MS based phosphoproteomics and dedicated bioinformatics. Expression of (phosphorylated)-EGFR was highly increased in EGFRvIII overexpressing cells and their secreted EVs. The increased phosphorylated proteins in both cells and EVs were associated with activated components of the EGFR-signalling cascade and included EGFR, AKT2, MAPK8, SMG1, MAP3K7, DYRK1A, RPS6KA3 and PAK4 kinases. In conclusion, EVs harbour oncogenic signalling networks including multiple activated kinases including EGFR, AKT and mTOR. SIGNIFICANCE: Extracellular vesicles (EVs) are biomarker treasure troves and are widely studied for their biomarker content in cancer. However, little research has been done on the phosphorylated protein profile within cancer EVs. In the current study, we demonstrate that EVs that are secreted by U87-EGFRvIII mutant glioblastoma cells contain high levels of oncogenic signalling networks. These networks contain multiple activated (phosphorylated) kinases, including EGFR, MAPK, AKT and mTOR.

DPHL: A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

Prostate Cancer Development Is Not Affected by Statin Use in Patients with Elevated PSA Levels.

Background: The role of statins in prostate cancer (PCa) remains unclear. Conflicting evidence has been found concerning risk reduction with the use of statins on biochemical recurrence (BCR). In this study, we evaluated whether statin use decreases the incidence of advanced PCa in males with elevated prostate-specific antigen (PSA; ≥4.0 ng/mL) levels and determined whether statin use reduces the risk of BCR after radical prostatectomy (RP). Methods: Patients visiting the outpatient urology clinic of the VU Medical Center between 2006 and 2018 with elevated PSA were retrospectively analyzed. Biochemical recurrence after RP was defined as a PSA level of ≥0.2 ng/mL (measured twice). Results: A total of 1566 patients were included, of which 1122 (72%) were diagnosed with PCa. At the time of diagnosis, 252 patients (23%) used statins compared to 83 patients (19%) in the non-malignancy group (p = 0.10). No differences were found in the use of statins between the different risk groups. No correlation was found between the risk of BCR after RP and the use of statins in the total (p = 0.20), the intermediate-risk group (p = 0.63) or the high-risk group (p = 0.14). Conclusion: The use of statins does not affect PCa development/progression in patients with elevated PSA levels, nor the development of BCR after RP.

Urinary exosomal proteins as (pan-)cancer biomarkers: insights from the proteome.

Exosomes are extracellular vesicles (EVs) released from cells under both physiological and pathological conditions, and may, thus, be present in biofluids. Urine is one of the most accessible biofluids implemented in clinical diagnostics. Recent mass spectrometry (MS)-based proteomic analyses have enabled high-throughput, deep proteome profiling of urinary EVs for the discovery, quantification and characterization of cancer-specific exosome biomarkers. The protein cargo of urine exosomes is emerging as an attractive source for biomarkers, not only for urological cancers, such as prostate, bladder and kidney cancer, but potentially also for nonurological cancers, including gastric, lung, oesophageal and colorectal cancer. More recently, exosome proteomics dissected protein cargo in the lumen and at the surface of EVs, and unexpectedly indicated that RNA- and DNA-binding proteins might also be present on vesicular surfaces. Here, we analyse MS-based proteomic data on urinary exosomes from cancer patients, and discuss the potential of urinary exosome-derived biomarkers in cancer.

Changes in the urinary extracellular vesicle proteome are associated with nephronophthisis-related ciliopathies.

Nephronophthisis is one of the leading genetic causes of end-stage renal disease in childhood. Early diagnostics and prognostics for nephronophthisis are currently limited. We aimed to identify non-invasive protein biomarkers for nephronophthisis in urinary extracellular vesicles. Extracellular vesicles were isolated from urine of 12 patients with a nephronophthisis-related ciliopathy and 12 age- and gender-matched controls, followed by in-depth label-free LC-MS/MS proteomics analysis of gel fractionated extracellular vesicle proteins. Supervised cluster analysis of proteomic profiles separated patients from controls. We identified 156 differentially expressed proteins with fold change ≥4 in patients compared to controls (P < .05). Importantly, expression levels of discriminating proteins were correlated with chronic kidney disease stage, suggesting possible applications for urinary extracellular vesicle biomarkers in prognostics for nephronophthisis. Enrichment analysis of gene ontology terms revealed GO terms including signaling, actin cytoskeleton and endocytosis among the downregulated proteins in patients, whereas terms related to response to wounding and extracellular matrix organization were enriched among upregulated proteins. Our findings represent the first step towards a non-invasive diagnostic test for nephronophthisis. Further research is needed to determine specificity of the candidate biomarkers. In conclusion, proteomic profiles of urinary extracellular vesicles differentiate nephronophthisis-related ciliopathy patients from healthy controls. SIGNIFICANCE: Nephronophthisis is an important cause of end-stage renal disease in children and is associated with an average diagnostic delay of 3.5 years. This is the first study investigating candidate biomarkers for nephronophthisis using global proteomics analysis of urinary extracellular vesicles in patients with nephronophthisis compared to control individuals. We show that measuring protein markers in urinary extracellular vesicles is a promising approach for non-invasive early diagnostics of nephronophthisis.

Feasibility of urinary extracellular vesicle proteome profiling using a robust and simple, clinically applicable isolation method.

Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultracentrifugation. The urinary EV isolation protocol using the Vn96-peptide is easier, time convenient (≈1.5 h) and no special equipment is needed, in contrast to ultracentrifugation protocol (>3.5 h), making this protocol clinically feasible. We compared the isolated vesicles of both ultracentrifugation and Vn96-peptide by proteome profiling using mass spectrometry-based proteomics (n = 4 per method). We reached a depth of >3000 proteins, with 2400 proteins that were commonly detected in urinary EVs from different donors. We show a large overlap (>85%) between proteins identified in EVs isolated by ultracentrifugation and Vn96-peptide. Addition of the detergent NP40 to Vn96-peptide EV isolations reduced levels of background proteins and highly increased the levels of the EV-markers TSG101 and PDCD6IP, indicative of an increased EV yield. Thus, the Vn96-peptide-based EV isolation procedure is clinically feasibly and allows large-scale protein profiling of urinary EV biomarkers.

The Non-Coding Transcriptome of Prostate Cancer: Implications for Clinical Practice.

Prostate cancer (PCa) is the most common type of cancer and the second leading cause of cancer-related death in men. Despite extensive research, the molecular mechanisms underlying PCa initiation and progression remain unclear, and there is increasing need of better biomarkers that can distinguish indolent from aggressive and life-threatening disease. With the advent of advanced genomic technologies in the last decade, it became apparent that the human genome encodes tens of thousands non-protein-coding RNAs (ncRNAs) with yet to be discovered function. It is clear now that the majority of ncRNAs exhibit highly specific expression patterns restricted to certain tissues and organs or developmental stages and that the expression of many ncRNAs is altered in disease and cancer, including cancer of the prostate. Such ncRNAs can serve as important biomarkers for PCa diagnosis, prognosis, or prediction of therapy response. In this review, we give an overview of the different types of ncRNAs and their function, describe ncRNAs relevant for the diagnosis and prognosis of PCa, and present emerging new aspects of ncRNA research that may contribute to the future utilization of ncRNAs as clinically useful therapeutic targets.

miR-129-3p controls centrosome number in metastatic prostate cancer cells by repressing CP110.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.

[18F]fluoromethylcholine as a chemotherapy response read-out in prostate cancer cells.

PURPOSE: The objective of the present study is to determine whether uptake of [(18)F]fluoromethylcholine ([(18)F]FCH) in comparison with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) accurately reflects chemotherapy efficacy at the tumor cell level in prostate cancer (PC). PROCEDURES: The effects of docetaxel and cabazitaxel on viable tumor cell number were explored in four PC cell lines. Cellular uptake of [(18)F]FDG and [(18)F]FCH was compared with the effects measured using sulforhodamine B (SRB) assay, cell counting and colony formation assay (CFA), as proximators of viable tumor cell number. Agreement between uptake and cell numbers was assessed by Bland-Altman plots. RESULTS: [(18)F]FCH uptake in all PC cell lines significantly correlated to the cell numbers surviving the respective drug concentrations. Bland-Altman analysis showed that [(18)F]FDG uptake resulted in signal overestimation and higher variability after chemotherapy. CONCLUSIONS: [(18)F]FCH uptake correlates well with viable tumor cell numbers remaining after docetaxel and cabazitaxel exposure. Radiolabeled choline is a potential response monitoring biomarker after chemotherapy for PC.

Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes.

Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3' end adenylated miRNAs are relatively enriched in cells, whereas 3' end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3' end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells.

PURPOSE: TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL. METHODS: Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms. RESULTS: The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression. CONCLUSIONS: TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.