Overcome Apoptosis Resistance Research Articles

Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Noxa/Bik Induction and Bcl-2/Bcl-xL Antagonism in Colon Cancer Cells.

KRAS mutations are frequently detected in human colorectal cancer and contribute to de novo apoptosis resistance and ultimately therapeutic failure. To overcome KRAS-mediated apoptosis resistance, the irreversible proteasome inhibitor, carfilzomib, was evaluated and found to potently induce Noxa, which was dependent upon c-Myc, and Bik. Isogenic mutant versus wild-type KRAS carcinoma cells showed elevated Bcl-xL, confirmed by KRAS siRNA or ectopic expression. Upregulated Bcl-xL by mutant KRAS was mediated by ERK as indicated by ERK knockdown. Bcl-xL expression was regulated at the level of mRNA and protein as shown using actinomycin D and cyclohexamide, respectively. Suppression of Bcl-xL by shRNA sensitized mutant KRAS cells to carfilzomib. Concurrent Bcl-xL antagonism by the BH3 mimetic ABT-263 combined with carfilzomib synergistically enhanced apoptosis that was dependent on Bax or p53, and was attenuated by Noxa or Bik shRNA. In support of this strategy, ectopically expressed Noxa enhanced apoptosis by ABT-263. Carfilzomib-induced Noxa and Bik sequestered Mcl-1 and ABT-263 released Bik and Bak from Bcl-xL, suggesting a mechanism for drug synergy. These preclinical findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance in KRAS-mutant colorectal cancer. Furthermore, antagonizing Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. This novel study reveals a promising treatment strategy to overcome apoptosis resistance in KRAS-mutant colorectal cancer by concurrent upregulation of Noxa/Bik and antagonism of Bcl-xL.

49 Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Bcl-xL Antagonism and Induction of Pro-Apoptotic BH3-Only Proteins in Colorectal Carcinoma Cells

Background. KRAS mutations, detected in at least 40% of human colorectal cancers (CRC), contribute to de novo apoptosis resistance and ultimately treatment failure. Aims. To study the mechanism of KRAS-mediated apoptosis resistance, we examined proand anti-apoptotic Bcl-2 family proteins in isogenic mutant vs wild type KRAS colorectal cancer (CRC) cells. Experimental Methods. Isogenic KRAS mutant CRC cells (HCT116, DLD1) and KRAS mutant SW620 cells were analyzed for Bcl-2 family proteins by immunoblotting. Cells were incubated with ABT-263 (1 μM), carfilzomib (0-50nM) or their combination; apoptosis was measured by annexin V+ labeling and caspase cleavage. Drug synergy was determined by calculation of a combination index. Protein-protein interaction was examined by immunoprecipitation. Knockdown of ERK or KRAS was achieved by siRNA. Ectopic expression of mutant KRAS or Noxa was achieved by a constitutive or doxycyline-inducible expression system, respectively. Results. We found that mutant KRAS cells showed upregulation of Bcl-xL that was attenuated by KRAS siRNA or reproduced by ectopic expression of mutant KRAS. Upregulation of Bcl-xL by mutant KRAS was mediated by ERK shown using EKR siRNA. Bcl-xL upregulation occurred at the level of mRNA and protein shown using actinomycin D and cyclohexamide, respectively. While BH3 mimetic agents can antagonize Bcl-2/BclxL, they are generally insufficient to overcome apoptosis resistance in solid tumors. Therefore, we evaluated a strategy of concurrent Bcl-xL antagonism and induction of pro-apoptotic BH3-only proteins. Carfilzomib, an irreversible proteasome inhibitor, was shown to induce Noxa and Bik proteins but failed to reverse mutant KRAS-mediated apoptosis resistance. However, concurrent antagonism of Bcl-xL by the BH3 mimetic, ABT-263, combined with carfilzomib was shown to synergistically enhance apoptosis in isogenic mutant KRAS CRC cell lines (Figure). In support of this strategy, we mimicked Noxa induction, seen with carfilzomib, using a doxycycline-inducible expression system. Ectopic Noxa was shown to markedly enhance apoptosis by ABT-263. A mechanism for drug synergy was suggested by the finding that Noxa and Bik proteins can sequester anti-apoptotic Mcl-1, while ABT-263 was able to release Bik and Bak from binding to Bcl-xL. Conclusion. Our novel findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance. Antagonism of Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Together, these preclinical data suggest a promising therapeutic strategy to overcome apoptosis resistance in KRAS mutant CRC cells.

Chk1 and Wee1 control genotoxic-stress induced G2–M arrest in melanoma cells

In the present report, the role of ATR–Chk1–Wee1 and ATM–Chk2–p53–p21 pathways in stress-induced cell cycle control is analysed in melanoma cells. Treatment of p53 wild-type melanoma cells with the genotoxic agent doxorubicin induces G2–M arrest, inhibitory phosphorylation of cell cycle kinase Cdc2 (CDK1) and enhanced expression of p53/p21. Wee1 inhibition under doxorubicin pulse-treatment reduces G2–M arrest and induces apoptosis. Inhibition of upstream kinase Chk1 under doxorubicin treatment almost completely abolishes stress-induced G2–M arrest and induces enhanced apoptosis. Interestingly, Chk1 inhibition alone even further increases apoptosis. While Chk1 inhibition alone almost completely abolishes G0–G1 arrest, combined treatment with doxorubicin re-establishes G0–G1 arrest. Moreover, Chk1 inhibition alone induces only a slight p53/p21 induction, while a strong induction of both proteins is observed by the combination with doxorubicin. These findings are suggestive for a particular role of p53/p21 in G0–G1, and Chk1 in G0–G1 and G2–M arrest. In line with this, the p53-mutant SK-Mel-28 melanoma cells do not mount a significant G0–G1 arrest under combined doxorubicin and Chk1 inhibitor treatment but rather show extensive apoptosis. Moreover, knockdown of p21 dramatically reduces stress-induced G0–G1 arrest under doxorubicin and Chk1 inhibitor treatment accompanied by massive DNA damage and apoptosis induction. Treatment of melanoma cells with an inhibitor of Chk2 upstream kinase ATM and doxorubicin almost completely abolishes G0–G1 arrest. Taken together, both Chk1 and Wee1 are mediators of G2–M arrest, while p53, p21 and Chk1 are mediators of G0–G1 arrest in melanoma cells. Combined treatment with chemotherapeutic agents such as doxorubicin and Chk1 inhibitors may help to overcome apoptosis resistance of p53-proficient melanoma cells. But treatment with Chk1 inhibitor alone may even be more efficient.

Serum vitamin D levels of patients with oral squamous cell carcinoma (OSCC) and expression of vitamin D receptor in oral precancerous lesions and OSCC.

Background: Resistance to programmed cell death (apoptosis) is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Vitamin D (calcitriol) may overcome apoptosis resistance in tumor cells of OSCC. Vitamin D receptor (VDR) expression in oral precancerous lesions of OSCC has not been analyzed and serum vitamin D level seems to be a predictor of cancer development. Material and Methods: Expression of VDR was analyzed in normal oral mucosa (n=5), oral precursor lesions (simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen (n=42) by immunohistochemistry (IHC). Moreover, serum vitamin D levels were measured by 25(OH)D3 (calcidiol) in patients with OSCC (n=42) and correlated with IHC results. Results: Expression of VDR was significantly increased in precancerous and OSCC compared with normal tissue. Compared with SIN I-III lesions VDR expression significantly decreased in OSCC. Severe vitamin D deficiency was detected in our OSCC patient cohort but there was no significant correlation analyzed between serum vitamin D levels and corresponding immunohistochemically detected VDR expression in OSCC. Conclusions: Our survey provides the first evidence of VDR expression in precancerous lesions of OSCC. Apoptosis induction of VDR+ cells in oral precancerous lesions and OSCC by natural vitamin D or synthetic vitamin D compounds could be useful for chemoprevention. Moreover, systemically and/or locally applied, these compounds may act as sensitizers for apoptosis mediated by radio-, and chemotherapy treatment in OSCC. Key words:Oral cancer, oral precancer, lichen planus, leukoplakia, apoptosis, serum 25(OH)D3, vitamin D receptor, chemoprevention, multistep carcinogenesis.

Abstract 2756: Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression

Abstract Sphingolipids are bioeffectors that mediate various cellular processes. We aimed to identify molecular targets of ceramide in the apoptosis pathway and to determine the efficacy of a ceramide analog in overcoming cancer cell resistance to apoptosis induction. We observed that exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to FasL-induced apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to FasL-induced apoptosis. LCL85 resembles Smac mimetic in sensitization of colon carcinoma cells to FasL-induced apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. Consistent with its apoptosis sensitization activity, LCL85 suppressed colon carcinoma cell metastatic potential in an experimental colon carcinoma lung metastasis mouse model. Taken together, we determined that a sublethal dose of LCL85 is effective as a sensitizer in overcoming apoptosis resistance of metastatic human colon carcinoma cells, and LCL85 is effective in suppressing tumor metastasis in vivo. Citation Format: Amy Paschall, Mary Zimmerman, Christina Torres, Dafeng Yang, May Chen, Xia Li, Erhard Bieberich, Aiping Bai, Jacek Bielawski, Alicja Bielawska, Kebin Liu. Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2014-2756

EGFR-targeted TRAIL and a Smac mimetic synergize to overcome apoptosis resistance in KRAS mutant colorectal cancer cells.

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed DbαEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of DbαEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward DbαEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of DbαEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing DbαEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects DbαEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-RasG12V. In the presence of doxycycline, these cells showed increased resistance to DbαEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the DbαEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between DbαEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that DbαEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.

Overcoming apoptosis resistance in high risk acute lymphoblastic leukemia by SMAC mimetics in a preclinical all xenograft model

Overcoming apoptosis resistance in high risk acute lymphoblastic leukemia by SMAC mimetics in a preclinical all xenograft model

Overcoming apoptosis resistance in high risk acute lymphoblastic leukemia by SMAC mimetics in a preclinical ALL xenograft model in vivo

Defects in cell death signaling e.g. overexpression of “Inhibitor of Apoptosis” (IAP) proteins are associated with poor prognosis and might be one reason for treatment failure and relapse of acute leukemia. Therefore, IAP antagonists, so called SMAC mimetics (SMs), provide a promising novel treatment strategy for pediatric ALL. In this study we investigated the effects of the small molecule SM BV6 on 42 primary ALL samples. Intriguingly, 70% of all individual patient-derived leukemias showed cell death induction after BV6 treatment in a TNF-α dependent manner. Previously, we described that rapid engraftment of ALL cells in NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in ALL cells and indicative for early patient relapse. Importantly, ALL samples with a TTLshort/early relapse phenotype showed activation of the constitutive deficient apoptosis signaling pathway upon BV6-treatment, demonstrating that SMs induce apoptosis signaling in former apoptosis resistant primary ALL cells. We further evaluated the in vivo efficacy of BV6 on high-risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. Most interestingly, a profound reduction of tumor load and prolonged survival of animals was observed upon BV6 in vivo treatment alone which was even more pronounced in combination with multidrug chemotherapy. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death inducing effect of BV6, indicating that BV6 induced apoptosis involves signaling via TNF-α and thereby provides a potential biomarker for the identification of patients who would benefit from SM treatment. Taken together, we show that the small molecule SM BV6 induces cell death via a TNF-α loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high-risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL.

Inhibitor of Apoptosis Proteins in Pediatric Leukemia: Molecular Pathways and Novel Approaches to Therapy

Inhibitor of Apoptosis (IAP) proteins are a family of proteins with antiapoptotic functions that contribute to the evasion of apoptosis, a form of programed cell death. IAP proteins are expressed at high levels in a variety of human cancers including childhood acute leukemia. This elevated expression has been associated with unfavorable prognosis and poor outcome. Therefore, IAP proteins are currently exploited as therapeutic targets for cancer drug discovery. Consequently, small-molecule inhibitors or antisense oligonucleotides directed against IAP proteins have been developed over the last years. Indeed, IAP antagonists proved to exhibit in vitro and in vivo antitumor activities against childhood pediatric leukemia in several preclinical studies. Thus, targeting IAP proteins represents a promising molecular targeted strategy to overcome apoptosis resistance in childhood leukemia, which warrants further exploitation.

The DeISGylase USP18 limits TRAIL-induced apoptosis through the regulation of TRAIL levels

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising molecule for anti-cancer therapies. Unfortunately, cancer cells frequently acquire resistance to rhTRAIL. Various co-treatments have been proposed to overcome apoptosis resistance to TRAIL. Here we show that downregulation of the deISGylase USP18 sensitizes cancer cells to rhTRAIL, whereas, elevate levels of USP18 inhibit TRAIL-induced apoptosis, in a deISGylase-independent manner. USP18 influences TRAIL signaling through the control of the IFN autocrine loop. In fact, cells with downregulated USP18 expression augment the expression of cellular TRAIL. Downregulation of cellular TRAIL abrogates the synergism between TRAIL and USP18 siRNA and also limits cell death induced by rhTRAIL. By comparing the apoptotic responsiveness to TRAIL in a panel of cancer cell lines, we have discovered a correlation between TRAIL levels and the apoptotic susceptibility to rhTRAIL, In cells expressing high levels of TRAIL-R2 susceptibility to rhTRAIL correlates with TRAIL expression. In conclusion, we propose that cellular TRAIL is an additional factor that can influence the apoptotic response to rhTRAIL.

Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Abstract Intensified treatment of pediatric acute lymphoblastic leukemia (ALL) has lead to increased survival rates of about 80%, however therapy fails in the remaining patients leading to relapse of the disease associated with inferior prognosis. Because treatment failure is, at least in part, due to defects in apoptosis programs, novel therapeutic strategies that counter apoptosis resistance are needed. “Inhibitor of Apoptosis” (IAP) proteins block the apoptosis machinery at a central point and are highly expressed in acute leukemias, thereby providing a target structure for therapeutic intervention. Molecules antagonizing these apoptosis inhibitors, so called SMAC-mimetics, therefore provide a promising strategy to overcome apoptosis deficiency and effectively treat high risk ALL. In this study, we investigated the effects of the small molecule SMAC-mimetic BV6 (kindly provided by Genentech) in B cell precursor- (BCP-) ALL. BV6 showed a clear induction of cell death at nanomolar concentrations in ALL cell lines. ALL cells sensitive for SMAC-mimetic induced cell death showed rapid cIAP degradation, NFkB activation and secretion of TNF-alpha (TNF-a). Interestingly, mitochondrial perturbation and caspase activation could be inhibited by the soluble TNF-a receptor Etanercept indicating the induction of a TNF-a dependent feed forward loop by the SMAC-mimetic BV6. In addition to cell lines, we investigated the effects of BV6 on a series of 42 primary ALL samples isolated from ALL bearing mice of established patient derived NOD/SCID/huALL xenograft leukemias. Intriguingly, upon treatment with the small molecule SMAC mimetic BV6, induction of cell death was observed in a majority of 70% of all individual patient-derived leukemias and BV6 induced cell death was inhibited by Etanercept demonstrating TNF-a dependency also in primary ALL. We previously described that rapid engraftment of ALL cells transplanted onto NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in the ALL cells and indicative for early patient relapse. Importantly, primary xenograft ALL samples with a TTLshort/early relapse phenotype showed increased cell death upon treatment with SMAC-mimetic BV6 and activation of the constitutive deficient apoptosis signaling pathway, demonstrating that SMAC-mimetics induce intact apoptosis signaling in former apoptosis resistant primary ALL cells. Based on theses findings, we further evaluated the in vivo effectivity of the SMAC-mimetic BV6 on high risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. ALL bearing recipients were treated with either BV6 or solvent for a given time of two weeks and further investigated for the presence of leukemia. Most interestingly, a significant delay of post-treatment leukemia reoccurrence was observed upon BV6 in vivo treatment along with a profound reduction of tumor load in the recipients compared to solvent treated animals. In a clinical setting, high-risk disease is unlikely to be treated by one compound alone. Therefore, we combined BV6 with multidrug chemotherapy resembling ALL induction treatment and observed a significant delay of ALL reoccurrence and prolonged survival of animals treated with the combination of the SMAC-mimetic and chemotherapy in contrast to chemotherapy alone. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death sensitizing effect of BV6, both in single treatment and in combination with chemotherapy. This indicates that BV6 induced apoptosis sensitization involves signaling via TNF-a and thereby provides a potential biomarker for the identification of patients who would benefit from SMAC-mimetic treatment. Taken together, we show that the small molecule SMAC-mimetic BV6 induces cell death via a TNF-a loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL. Thus, induction of cell death by new generation small molecule SMAC-mimetics provides a promising novel strategy for targeted therapy of high-risk acute lymphoblastic leukemia and involvement of TNF-a signaling in BV6-sensitive patients points to its potential use as biomarker indicating effective cell death sensitization. Disclosures: No relevant conflicts of interest to declare.

Smac mimetic primes apoptosis-resistant acute myeloid leukaemia cells for cytarabine-induced cell death by triggering necroptosis

The prognosis for patients with acute myeloid leukaemia (AML) is still poor, thus calling for novel treatment strategies. Here, we report that the small-molecule Smac mimetic BV6, which antagonizes Inhibitor of Apoptosis (IAP) proteins, acts in concert with cytarabine (AraC) to trigger cell death in AML cells in a highly synergistic manner (combination index 0.02–0.27). Similarly, BV6 cooperates with AraC to trigger cell death in primary AML samples, underscoring the clinical relevance of our findings. Molecular studies reveal that the TNFα-blocking antibody Enbrel significantly reduces BV6/AraC-induced cell death, demonstrating that an autocrine/paracrine TNFα loop mediates cell death. Furthermore, BV6 and AraC synergize to induce loss of mitochondrial membrane potential, caspase activation and DNA fragmentation, consistent with apoptotic cell death. Nevertheless, the caspase inhibitor zVAD.fmk fails to protect against BV6/AraC-induced cell death. Intriguingly, this cell death upon caspase inhibition is significantly reduced by pharmacological inhibition of two key components of necroptosis signaling, i.e. by RIP1 kinase inhibitor Necrostatin-1 or MLKL inhibitor NSA. Thus, BV6 sensitizes AML cells to AraC-induced cell death and overcomes apoptosis resistance by triggering necroptosis as alternative form of cell death. These findings have important implications for Smac mimetic-based strategies to bypass apoptosis resistance of AML.

Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis

Evasion of apoptosis, for example, by inhibitor of apoptosis (IAP) proteins, contributes to treatment resistance and poor outcome in acute myeloid leukemia (AML). Here we identify a novel synergistic interaction between the small-molecule second mitochondria-derived activator of caspases (Smac) mimetic BV6, which antagonizes X-linked IAP, cellular IAP (cIAP)1 and cIAP2, and the demethylating agents 5-azacytidine or 5-aza-2′-deoxycytidine (DAC) to induce cell death in AML cells, including apoptosis-resistant cells. Calculation of combination index (CI) confirms that this drug combination is highly synergistic (CI 0.02–0.4). In contrast, BV6 and DAC at equimolar concentrations do not cause synergistic toxicity against normal peripheral blood lymphocytes, pointing to some tumor cell selectivity. Molecular studies reveal that BV6 and DAC cooperate to trigger the activation of caspases, mitochondrial perturbations and DNA fragmentation, consistent with apoptotic cell death. However, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to protect against BV6/DAC-induced cell death and even significantly increases the percentage of Annexin-V/propidium iodide double-positive cells. Importantly, BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. This indicates a switch from BV6/DAC-induced cell death from apoptosis to necroptosis upon caspase inhibition. Thus, BV6 cooperates with demethylating agents to induce cell death in AML cells and circumvents apoptosis resistance via a switch to necroptosis as an alternative mode of cell death. The identification of a novel synergism of BV6 and demethylating agents has important implications for the development of new treatment strategies for AML.

Melatonin overcomes apoptosis resistance in human hepatocellular carcinoma by targeting Survivin and XIAP

Apoptosis resistance in hepatocellular carcinoma (HCC) is one of the most significant factors for hepatocarcinogenesis and tumor progression, and leads to resistance to conventional chemotherapy. It is well known that inhibitor of apoptosis proteins (IAPs) play key roles in apoptosis resistance, it has become an important target for antitumor therapy. In this study, we examined if melatonin, the main secretory product of the pineal gland, targeted IAPs, leading to the inhibition of apoptosis resistance. To accomplish this, we first observed that four members of IAPs (cIAP-1, cIAP-2, survivin, and XIAP) were overexpressed in human HCC tissue. Interestingly, melatonin significantly inhibited the growth of HepG2 and SMMC-7721 cells and promoted apoptosis along with the downregulation of survivin and XIAP, but had no effect on the expression of cIAP-1 and cIAP-2. These data suggest that the inhibition of survivin and XIAP by melatonin may play an important part in reversing apoptosis resistance. Notably, cIAP-1, survivin and XIAP were significantly associated with the coexpression of COX-2 in human HCC specimens. Melatonin also reduced the expression of COX-2 and inhibited AKT activation in HepG2 and SMMC-7721 cells. Inhibition of COX-2 activity with the selective inhibitor, NS398, and inhibition of AKT activation using the PI3K inhibitor, LY294002, in tumor cells confirmed that melatonin-induced apoptosis was COX-2/PI3K/AKT-dependent, suggesting that the COX-2/PI3K/AKT pathway plays a role in melatonin inhibition of IAPs. Taken together, these results suggest that melatonin overcomes apoptosis resistance by the suppressing survivin and XIAP via the COX-2/PI3K/AKT pathway in HCC cells.

MicroRNA-24 regulates XIAP to reduce the apoptosis threshold in cancer cells

MicroRNAs have been implicated as important mediators of cancer cell homeostasis, and accumulating data suggest compelling roles for them in the apoptosis pathway. X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor and an important barrier to apoptotic cell death, but the mechanisms which determine the diverse range of XIAP expression seen in cancer remains unclear. In this study, we present evidence that miR-24 directly targets the 3′UTR of the XIAP mRNA to exert translational repression. Using a heuristic algorithm of bioinformatics analysis and in vitro screening, we identified miR-24 as a candidate regulator of XIAP expression. Array CGH and SKY analysis reveal that genomic copy number loss at the miR-24 locus is concordant with loss of endogenous miR-24 in cancer cells. Using a luciferase construct of the XIAP 3′UTR, we showed that miR-24 specifically coordinates to the XIAP mRNA. And interference with miR-24’s binding of the critical seed region, resulting from site-directed mutagenesis of the 3′UTR, significantly abrogated miR-24’s effects on XIAP expression. Moreover, miR-24 over-expression can overcome apoptosis-resistance in cancer cells via down-regulation of XIAP expression, and the resulting cancer cell death induced by TRAIL is executed by the canonical caspase-mediated apoptosis pathway. In summary, our data suggest a novel mechanism by which miR-24 directly modulates XIAP expression level and consequently the apoptosis threshold in cancer cells.

Arginylated Calreticulin at Plasma Membrane Increases Susceptibility of Cells to Apoptosis

Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a preapoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase are less susceptible to apoptosis than wild type cells. Under these conditions R-CRT is present on the cell membrane but at early stages is differently localized in stress granules. Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able to both increase the number of cells undergoing apoptosis in wild type cells and overcome apoptosis resistance in cells lacking arginyl-tRNA protein transferase that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function, and survival.

Abstract 2278: Smac mimetic primes FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis and overcomes apoptosis resistance

Abstract Evasion of apoptosis contributes to treatment resistance, one of the major, yet unresolved obstacles in oncology. Searching for new strategies to bypass apoptosis resistance we investigated the potential of Smac mimetic in acute lymphoblastic leukemia (ALL) cells deficient in FADD or caspase-8. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant leukemia cells for TNFα-induced necroptosis as an alternative mode of cell death. The interaction of Smac mimetic and TNFα occurs is highly synergistic as calculated by combination index (CI <0.1). Kinetic analysis reveals that Smac mimetic and TNFα cause cell death rapidly within few hours. Interestingly, Smac mimetic- and TNFα-mediated cell death in FADD-deficient cells occurs without characteristic features of apoptosis, namely without caspase activation or DNA fragmentation, pointing to non-apoptotic cell death. In sharp contrast, Smac mimetic and TNFα trigger activation of caspase-8, -9 and -3 and DNA fragmentation in apoptosis-proficient wildtype cells that express FADD and caspase-8. Consistently, the caspase inhibitor zVAD.fmk protects wildtype cells against Smac mimetic- and TNFα-triggered cell death, whereas zVAD.fmk fails to block cell death in FADD-deficient cells. Furthermore, production of reactive oxygen species (ROS) precedes Smac mimetic- and TNFα-induced cell death in FADD-deficient cells. In line with the notion that ROS contributes to cell death induction, the addition of ROS scavengers (i.e. butylated hydroxyanisole (BHA) and trolox) or the addition of the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduce cell death upon combination treatment. Moreover, Smac mimetic- and TNFα-triggered cell death is accompanied by enhanced RIP1 kinase activity in FADD-deficient cells. Of note, necrostatin-1, a RIP1 kinase inhibitor, reduces Smac mimetic- and TNFα-induced ROS generation and completely abolishes cell death in FADD-deficient cells, in accordance with necroptotic cell death. Importantly, Smac mimetic also sensitizes apoptosis-resistant, primary leukemic blasts derived from patients with ALL to TNFα-induced necroptosis, underscoring the clinical relevance of these findings. In conclusion, Smac mimetic primes leukemia cells to TNFα-induced apoptosis or necroptosis in a context-dependent manner. In apoptosis-proficient cells which express both FADD and caspase-8, Smac mimetic enhances TNFα-induced, caspase-dependent apoptosis. However in apoptosis-resistant cells lacking either FADD or caspase-8, Smac mimetic sensitizes cells for TNFα-initiated necroptosis that critically depends on RIP1 and ROS production. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2278. doi:1538-7445.AM2012-2278

Stimulation of the cytosolic dsRNA sensor MDA-5 induces cell death in fibroblast-like synoviocytes

Background and objectivesRheumatoid arthritis is characterised by hyperplasia of the synovial tissue due to local proliferation of stromal and recruitment of inflammatory immune cells. Accumulation of synovial fibroblasts likely is...

Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1

The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas.

Leflunomide Induces Apoptosis in Fludarabine-Resistant and Clinically Refractory CLL Cells

Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4-induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4-activated ("resistant") CLL cells was achieved with higher concentrations (IC(50): 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC(50): 56 μg/mL). We thus show that A771726 overcomes CD40L/IL-4-mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells.