Kaamar Azijli

Non-canonical kinase signaling by the death ligand TRAIL in cancer cells: discord in the death receptor family

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based therapy is currently evaluated in clinical studies as a tumor cell selective pro-apoptotic approach. However, besides activating canonical caspase-dependent apoptosis by binding to TRAIL-specific death receptors, the TRAIL ligand can activate non-canonical cell survival or proliferation pathways in resistant tumor cells through the same death receptors, which is counterproductive for therapy. Even more, recent studies indicate metastases-promoting activity of TRAIL. In this review, the remarkable dichotomy in TRAIL signaling is highlighted. An overview of the currently known mechanisms involved in non-canonical TRAIL signaling and the subsequent activation of various kinases is provided. These kinases include RIP1, IκB/ NF-κB, MAPK p38, JNK, ERK1/2, MAP3K TAK1, PKC, PI3K/Akt and Src. The functional consequences of their activation, often being stimulation of tumor cell survival and in some cases enhancement of their invasive behavior, are discussed. Interestingly, the non-canonical responses triggered by TRAIL in resistant tumor cells resemble that of TRAIL-induced signals in non-transformed cells. Better knowledge of the mechanism underlying the dichotomy in TRAIL receptor signaling may provide markers for selecting patients who will likely benefit from TRAIL-based therapy and could provide a rationalized basis for combination therapies with TRAIL death receptor-targeting drugs.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3 dependent invasion in resistant non-small cell lung cancer cells

Summary Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.

Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells

Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC-MS/MS which sugars accumulated, their subcellular localization (using (3)H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with (3)H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane. Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis.

Cross-resistance to clinically used tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib

PurposeWhen during cancer treatment resistance to a tyrosine kinase inhibitor (TKI) occurs, switching to another TKI is often considered as a reasonable option. Previously, we reported that resistance to sunitinib may be caused by increased lysosomal sequestration, leading to increased intracellular lysosomal storage and, thereby, inactivity. Here, we studied the effect of several other TKIs on the development of (cross-) resistance.MethodsTKI resistance was induced by continuous exposure of cancer cell lines to increasing TKI concentrations for 3–4xa0months. (Cross-) resistance was evaluated using MTT cell proliferation assays. Intracellular TKI concentrations were measured using LC-MS/MS. Western blotting was used to detect lysosome-associated membrane protein-1 and −2 (LAMP1/2) expression.ResultsThe previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib. Exposure of 786-O and HT-29 cells to sorafenib, pazopanib or erlotinib for 3–4xa0months induced drug resistance to pazopanib and erlotinib, but not sorafenib. Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells. Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient. No cross-resistance to the mTOR inhibitor everolimus was detected.ConclusionsOur data indicate that tumor cells can develop (cross-) resistance to TKIs, and that such resistance includes increased intracellular drug accumulation accompanied by increased lysosomal storage. Transient (cross-) resistance was found to occur for several of the TKIs tested, but not for everolimus, indicating that switching from a TKI to a mTOR inhibitor may be an attractive therapeutic option.

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

PurposeTRAIL, 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.MethodsInteractions 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.ResultsThe 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 24xa0h followed by 48xa0h 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.ConclusionsTFT 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.

Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer

Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5–10u2009μg of lysate protein and up to 400u2009μM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5–7u2009μg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides.

The Potential Role of Lysosomal Sequestration in Sunitinib Resistance of Renal Cell Cancer

Renal cell carcinoma (RCC) is a highly vascularized tumor type, which is often associated with inactivated mutations in the von Hippel-Lindau gene that drives proangiogenic signaling pathways. As such, new therapies for the treatment of RCC have largely been focused on blocking angiogenesis. Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the most frequently used first-line drug for the treatment of RCC. Although treatment with sunitinib improves patient outcome considerably, acquired resistance will emerge in all cases. The molecular mechanisms of resistance to sunitinib are poorly understood, but in the past decade, several of these have been proposed. Lysosomal sequestration of sunitinib was reported as a potential resistance mechanism to sunitinib. In this review, the underlying molecular mechanisms of lysosomal sunitinib sequestration and the potential strategies to overcome this resistance are discussed to be able to further improve the treatment of RCC.

Abstract 1941: TRAIL-induced kinase activation in Non small cell lung cancer cells

Non-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel therapeutic approaches are greatly needed. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an interesting agent that is able to trigger apoptosis through interactions with TRAIL receptors. An important feature of TRAIL is its ability to induce apoptosis in a wide variety of tumors without harming normal cells, making it an attractive anti-cancer therapeutic compared to conventional anti-cancer agents. However, TRAIL is also able to activate signaling pathways that are involved in survival, proliferation and migration of tumor cells. Thus, TRAIL-based therapies combined with inhibitors of such pathways are expected to enhance therapeutic benefit. In this study, we aimed to identify and characterize kinases that are activated by recombinant TRAIL in NSCLC cells. We monitored the activation of a number of kinases known to be involved in TRAIL signaling by Western blotting, including p38 MAPK, JNK, ERK and Akt. In addition we employed PepChip kinase arrays. With these arrays 1024 peptide kinase substrates can be screened in one experiment, whereby a comparison of kinase patterns between untreated and treated cells can be obtained. NSCLC, H460 and A549 cell lines, which are sensitive and resistant for TRAIL, respectively, were exposed to 50 ng/ml TRAIL for different periods of time (5 to 240 minutes) to evaluate the kinetics of kinase activation. In H460 cells, TRAIL induced the phosphorylation of p38 MAPK after 2 hours and JNK1/2 after 3 hours. As the activation of these kinases can be both anti- and pro-apoptotic, kinase inhibitors were used to explore this further. In H460 cells the activation of JNK, ERK and Akt had anti-apoptotic activity. Inhibition of these kinases with SP600125, PD098059, and LY294002, respectively, showed a 2-fold increase in apoptosis when combined with TRAIL. The activation of MAPK p38 on the other hand was pro-apoptotic, since its inhibition with SB203580 resulted in a reduction of TRAIL-induced apoptosis in H460 cells. In resistant A549 cells, however, Akt, ERK, p38 MAPK, and JNK1/2 activation appeared to have anti-apoptotic activity. Furthermore, in these cells an increase in IκBα phosphorylation was observed that was not seen in H460 cells, where levels of phosphorylated IκBα decreased after 1 hour that correlated with cleavage of RIP. Thus suppression of NFκB activation could be associated with TRAIL sensitivity. PepChip kinase arrays, revealed the activation of kinases that are involved in cell migration, such as Rho/Rock in A549 cells, and further investigations are ongoing. In conclusion, we observed differential TRAIL-dependent activation of p38 MAPK, JNK1/2, ERK, Akt and IκBα in sensitive and resistant NSCLC cells as well as in pathways that regulate migration. The relationship with TRAIL antitumor activity is currently further explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2011-1941

Abstract 1261: TRAIL-induced pro- and antiapoptotic kinase activation in non-small cell lung cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnNon-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel therapeutic approaches are greatly needed. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to the TNF gene superfamily and it mediates its apoptotic function through interaction with the death receptors, DR4 and DR5. An important feature of TRAIL is its ability to induce apoptosis in a wide variety of tumors without harming normal cells, making it an attractive anti-cancer therapeutic compared to conventional anti-cancer agents. However, TRAIL is also able to activate signaling pathways that are implicated in cell survival and cell proliferation, counteracting its death-inducing effects. Thus, TRAIL-based therapies combined with inhibitors of pro-survival signals are expected to have beneficial therapeutic effects.nnIn this study, we examined the pro-survival signaling induced by recombinant TRAIL in NSCLC cells. We monitored the activation of a number of kinases known to be involved in TRAIL signaling by Western blotting, including p38 MAPK, JNK, ERK and Akt. In addition we employed PepChip kinase arrays. With these arrays 1024 peptide kinase substrates can be screened in one experiment, whereby a comparison of kinase patterns between untreated and treated cells can be obtained.nnH460 and A549 NSCLC cell lines, which are sensitive and resistant for TRAIL, respectively, were exposed to 50 ng/ml TRAIL for different periods of time (5, 10, 15, 30, 60, 120, 180, 240 minutes) to evaluate the kinetics of the kinases. In H460 cells, TRAIL phosphorylated p38 MAPK after 2 hours and JNK1/2 after 3 hours. As the activation of these kinases can be both anti- and pro-apoptotic, kinase inhibitors were used to explore this further. In H460 cells the activation of JNK, ERK and Akt had anti-apoptotic activity. Inhibition of these kinases with SP600125, PD098059, and LY294002, respectively, showed a 2 fold increase in apoptosis when combined with TRAIL. The activation of MAPK p38 on the other hand was pro-apoptotic, since its inhibition with SB203580 resulted in a reduction of TRAIL-induced apoptosis in H460 cells. In resistant A549 cells, however, Akt, ERK, p38 MAPK, and JNK1/2 activation appeared to have anti-apoptotic activity. Furthermore, in these cells an increase in IκBα phosphorylation was observed that was not seen in H460 cells, where levels of phosphorylated IκBα decreased after 1 hour that correlated with cleavage of RIP. Thus reduction of NFκB activation could be associated with TRAIL sensitivity. Additional experiments, including the PepChip kinase arrays, are ongoing to further examine the involvement of these and other kinases in TRAIL sensitivity and for possible use in sensitization strategies. In conclusion, we observed differential TRAIL-dependent activation of p38 MAPK, JNK1/2, ERK, Akt and IκBα in sensitive and resistant NSCLC cells and the relationship with TRAIL sensitivity is currently further explored.nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1261.