Frank A.E. Kruyt

Cell Death Independent of Caspases: A Review

Patterns of cell death have been divided into apoptosis, which is actively executed by specific proteases, the caspases, and accidental necrosis. However, there is now accumulating evidence indicating that cell death can occur in a programmed fashion but in complete absence and independent of caspase activation. Alternative models of programmed cell death (PCD) have therefore been proposed, including autophagy, paraptosis, mitotic catastrophe, and the descriptive model of apoptosis-like and necrosis-like PCD. Caspase-independent cell death pathways are important safeguard mechanisms to protect the organism against unwanted and potential harmful cells when caspase-mediated routes fail but can also be triggered in response to cytotoxic agents or other death stimuli. As in apoptosis, the mitochondrion can play a key role but also other organelles such as lysosomes and the endoplasmic reticulum have an important function in the release and activation of death factors such as cathepsins, calpains, and other proteases. Here we review the various models of PCD and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of caspase-independent cell death pathways for cancer.

Global Histone Modifications Predict Prognosis of Resected Non–Small-Cell Lung Cancer

PURPOSE Epigenetic modifications may contribute to the development and progression of cancer. We investigated whether epigenetic changes involving multiple histones influence prognosis of non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS We used immunohistochemistry to assess histone 3 lysine 4 dimethylation (H3K4diMe), and acetylation of histone 2A lysine 5 (H2AK5Ac), histone 2B lysine 12, histone 3 lysine 9 (H3K9Ac), and histone 4 lysine 8 in resected tumor samples of 138 NSCLC patients. Data were analyzed using a recursive partitioning analysis (RPA). RESULTS The RPA classified the patients into seven distinct prognostic groups based on TNM stage (first node), histology, and histone modifications: H3K4diMe (< or 85% tumor cells), H3K9Ac (< or 68% tumor cells), and H2AK5Ac (< or 5% tumor cells). The seven groups were associated with significantly different disease-free (P < .0001) and overall survival (P < .0001). Interestingly, the four groups determined by stage I patients (below the first node) displayed dramatic differences in survival (median, 10 months in adenocarcinoma patients with H3K9Ac 68% v 147 months in nonadenocarcinoma patients with H3K4diMe 85%). A Cox model retained age and RPA groups as the sole independent factors significantly influencing overall survival. CONCLUSION The prognostic influence of epigenetic changes involving multiple histones, in particular H2A and H3, is greater in early NSCLC, and evaluation of these changes may help in selecting early-stage NSCLC patients for adjuvant treatment. Our observations provide a rationale for the use of a combination of standard chemotherapy with drugs interacting with histone modifications, such as histone deacetylase inhibitors.

Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells.

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.

TRAIL and cancer therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors are promising targets for the selective eradication of tumor cells while sparing normal cells. Currently, both recombinant TRAIL proteins and TRAIL receptor agonistic antibodies are being tested in the clinic, showing encouraging antitumor activities and mild side effects. Unfortunately, resistance to TRAIL therapy is frequently encountered requiring combined treatments with sensitizing agents. Standard chemotherapeutics can enhance TRAIL sensitivity; however, more specific and less toxic agents are needed to exploit the full antitumor potential of TRAIL. Here, a brief overview of the TRAIL signaling pathway is given together with a short description of early results obtained with TRAIL therapy in the clinic. Mechanisms of TRAIL resistance and ways to overcome these by targeted agents that either neutralize apoptotic blockades or suppress prosurvival signals also triggered by TRAIL are highlighted, such as inhibitors of IAPs, Bcl-2 family members, HDACi, and modulators of NF-kappaB, Raf and EGFR signaling.

Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells

In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k‐ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib‐sensitivity in egfr wild type cells. Activating k‐ras mutations were specifically detected in gefitinib‐resistant cells, suggesting that the occurrence of k‐ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib‐sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib‐resistance of NSCLC cell lines. Gefitinib‐resistant NSCLC cell lines, showing EGFR‐independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co‐treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy.

Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.

Circulating tumor cells in small-cell lung cancer: a predictive and prognostic factor

BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. Patients and methods In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14,040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. PATIENTS AND METHODS In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch® systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14 040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.

Toward a new generation of conditionally replicating adenoviruses : Pairing tumor selectivity with maximal oncolysis

Conditionally replicating adenoviruses (CRADs) represent a promising new platform for the treatment of cancer. CRADs have been demonstrated to kill tumor cells when other therapies fail, indicating that their antitumor properties are complementary to, and distinct from, those of standard treatments such as chemotherapy and radiation. In clinic trials CRADs have shown encouraging results, demonstrating mild side effects when administered at high doses and via different routes, including intratumorally, intraperitoneally, and intravenously. Tumor-selective replication has been detected, although as a single agent the efficacy appears to be limited. Interestingly, combined treatment with radiation or chemotherapy has been found to enhance CRAD efficacy considerably. To date, the molecular mechanisms underlying adenovirus-mediated oncolysis, and the way in which chemotherapy enhances oncolysis, are not well understood. A fuller knowledge of these processes will open up new strategies to improve the cell-killing potential of CRADs. Here, we discuss several possibilities that may lead to CRADs with enhanced oncolytic activity. These approaches include strategies to functionally couple tumor targeting and optimal oncolytic activity, and ways to further increase tumor cell disruption at later stages of infection to facilitate the spreading of virus throughout the tumor mass. In addition, improved methods to evaluate the efficacy of these agents in animal models, and in the clinic, will be required to systematically test and optimize CRAD efficacy, also taking into account the influence of tumor characteristics and the administration route.

TRAIL therapy in non-small cell lung cancer cells: sensitization to death receptor mediated apoptosis by proteasome inhibitor bortezomib

Activation of the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor pathway is a promising therapeutic strategy to selectively eradicate cancer cells, including non–small cell lung cancer (NSCLC) cells. Recombinant human (rh) TRAIL/Apo-2L, a TRAIL-encoding adenovirus, and monoclonal antibodies directed against TRAIL receptors R1 and R2 were used to study cytotoxicity of TRAIL therapy in NSCLC cells. NSCLC cells showed differential sensitivity to TRAIL therapy, regardless of the agent used. Combination treatment of bortezomib and rhTRAIL led to synergistic apoptosis induction in NSCLC cell lines. Enhancement of rhTRAIL-induced apoptosis by bortezomib was caspase dependent, implicating extrinsic as well as intrinsic apoptosis activation, as shown by increased processing of caspase-8 as well as caspase-9, and could be abrogated completely by overexpression of caspase-8 inhibitor cytokine response modifier A (CrmA), and partially by overexpression of Bcl-2. Enhanced surface expression of TRAIL-R2, but also TRAIL-R1, was associated with bortezomib treatment, which is likely to contribute to the increased processing of caspase-8 in the combination treatment. Furthermore, TRAIL-induced activation of prosurvival transcription factor nuclear factor-κB was prevented by cotreatment with bortezomib, which may contribute to the observed synergistic apoptosis induction. Our preclinical data indicate that combination therapy of TRAIL and bortezomib may be an effective strategy for NSCLC. [Mol Cancer Ther 2007;6(7):2103–12]

Assessment of IAP (inhibitor of apoptosis) proteins as predictors of response to chemotherapy in advanced non-small-cell lung cancer patients

BACKGROUND Expression of inhibitor of apoptosis family proteins (IAPs) has been shown in vitro to decrease chemosensitivity through caspase inhibition. However, the role of IAPs as predictors of response to chemotherapy in cancer patients remains to be determined. PATIENTS AND METHODS Using immunohistochemistry, we assessed the expression of the IAP proteins c-IAP1, c-IAP2, and XIAP on tumors from 55 patients with advanced non-small-cell lung cancer (NSCLC) treated with chemotherapy, and correlated that with the observed response to chemotherapy, time to progression and overall survival. RESULTS Differences were observed in the pattern of staining among the IAP proteins. The expression of c-IAP2 and XIAP was exclusively cytoplasmic. whereas c-IAP1 also displayed nuclear staining. The median expression of tumor cells for c-IAP1, c-IAP2, and XIAP was 70%, 45%, and 25%, respectively, and a correlation was observed between c-IAP1 and c-IAP2 (P = 0.004), and c-IAP1 and XIAP expression (P = 0.013). However, no association was seen between the expression of these proteins and sex, age, tumor size, stage, histology and grade of differentiation. Interestingly, expression of c-IAP1, c-IAP2, and XIAP did not predict response to chemotherapy. In addition, the expression of IAPs had no impact on the time to progression or overall survival of this group of patients. CONCLUSIONS Our results indicate that: 1) there are differences in the level of expression and in the subcellular distribution of c-IAP1, c-IAP2, and XIAP in tumors derived from NSCLC patients. 2) The expression of c-IAP1, c-IAP2 and XIAP does not predict the response to chemotherapy in patients with advanced NSCLC. 3) The relation between expression of IAPs and chemosensitivity in cancer patients may be more complex than anticipated by in vitro data.