Pancreatic Cancer Chemoresistance by Inhibition of TAK1
Pancreatic Cancer Chemoresistance by Inhibition of TAK1
Background TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer.
Methods NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided.
Results AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC50 between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029).
Conclusions The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.
Pancreatic adenocarcinoma is one of the most lethal and poorly understood human malignancies. Because of the lack of effective systemic therapies, the 5-year survival rate for patients with pancreatic adenocarcinoma has remained at 1%–3%, without change over the past 25 years. Hence, development of novel chemotherapeutic approaches that reduce the intrinsic drug resistance of this disease poses one of the greatest challenges in pancreatic cancer research.
Nuclear factor κB (NF-κB) and activator protein-1 (AP-1) are key transcriptional factors that orchestrate expression of many genes involved in inflammation, oncogenesis, and apoptosis. NF-κB is constitutively activated in numerous hematologic malignancies and solid tumors, including pancreatic cancer, and its activation can suppress proapoptotic signaling pathways through the expression of several antiapoptotic genes. The exact function of AP-1 in cellular responses to genotoxic stress has not been completely elucidated, but it could be associated with the concomitant activation of other pathways known to mediate survival, including NF-κB. In particular, Lamb et al. demonstrated that the AP-1 transcription factor JunD cooperates with NF-κB to increase the expression of prosurvival genes that contain both NF-κB- and AP-1-binding sites in their promoters. Because much of the cytotoxicity of chemotherapeutic agents occurs through apoptosis, the coactivation of NF-κB and AP-1, which can synergistically and effectively suppress the apoptotic potential of chemotherapeutic agents, could be a crucial obstacle to effective treatment of cancer
We recently demonstrated that an autocrine stimulation of interleukin 1 alpha (IL-1α), primarily mediated through induction of AP-1 activity, accounted for the constitutive activation of NF-κB and thus for the metastatic behavior of pancreatic cancer. During immune and inflammatory responses, detailed investigation of IL-1-induced tumor necrosis factor (TNF) receptor associated factor (TRAF)-6 signaling demonstrated activation of NF-κB through two parallel signaling pathways, depending on differential activation of two mitogen-activated protein kinase kinase kinases (MAP3Ks), MEKK3 (MAP3K3), or the TGF-β-activated kinase-1 (TAK1; MAP3K7).
TAK1 was originally identified as a MAP3K, which can be rapidly activated in response to TGF-β signal transduction. In vitro studies have demonstrated that overexpression of a dominant negative version of TAK1 inhibits both the activation of NF-κB and the mediator of AP-1 induction, c-Jun N-terminal kinase (JNK), thus increasing the sensitivity of cells to apoptosis induced by TNF-α. Mice carrying an epidermal-specific deletion of the TAK1 gene developed severe skin inflammation caused by impaired activation of NF-κB and JNK in response to TNF, which resulted in a massive apoptosis of keratinocytes much greater than those observed in IκB kinase beta (IKKβ) and IKKγ deletion models. A mouse model with TAK1 conditionally deleted in T cells was used to demonstrate that TAK1 is essential for in vivo thymocyte development and activation. The loss of TAK1 in the thymocytes prevented the activation of IKK, NF-κB, and JNK and sensitized the mutant cells to activation-induced apoptosis. Using a B cell-conditional TAK1-deficient mouse model, Sato et al. demonstrated that TAK1 is essential for toll-like receptor, IL-1 receptor, TNF receptor, and B cell receptor cellular responses and signaling pathways leading to the activation of JNK and/or NF-κB. Suppression of TAK1 signaling by dominant negative TAK1 reduced NF-κB activation in human head and neck squamous cell carcinoma and breast cancer cell lines.
Cellular inhibitor of apoptosis 2 (cIAP-2) is a member of the inhibitor of apoptosis (JAP) family of proteins that regulate programmed cell death by directly inhibiting caspases and by targeting proapoptotic components of the TNF-α signaling pathways for ubiquitin degradation. The overexpression of cIAP-2 is a common and early event in the progression of pancreatic cancer. Although the expression of cIAP-1 is constantly high in both normal and neoplastic pancreatic tissues, cIAP-2 mRNA levels are statistically significantly higher in pancreatic cancer than in normal pancreatic tissue. A sequence analysis of the cIAP-2 promoter revealed two critical NF-κB-binding sites and two potential AP-1-binding sites.
We hypothesized that TAK1 might be responsible for the resistance of pancreatic cancer to the proapoptotic effect of chemotherapeutic agents by increasing the NF-κB- and AP-1-mediated transcription of cIAP-2. Thus, targeting the expression or the kinase activity of TAK1 might reverse the intrinsic resistance of pancreatic cancer to chemotherapy.
Abstract and Introduction
Abstract
Background TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer.
Methods NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided.
Results AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC50 between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029).
Conclusions The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.
Introduction
Pancreatic adenocarcinoma is one of the most lethal and poorly understood human malignancies. Because of the lack of effective systemic therapies, the 5-year survival rate for patients with pancreatic adenocarcinoma has remained at 1%–3%, without change over the past 25 years. Hence, development of novel chemotherapeutic approaches that reduce the intrinsic drug resistance of this disease poses one of the greatest challenges in pancreatic cancer research.
Nuclear factor κB (NF-κB) and activator protein-1 (AP-1) are key transcriptional factors that orchestrate expression of many genes involved in inflammation, oncogenesis, and apoptosis. NF-κB is constitutively activated in numerous hematologic malignancies and solid tumors, including pancreatic cancer, and its activation can suppress proapoptotic signaling pathways through the expression of several antiapoptotic genes. The exact function of AP-1 in cellular responses to genotoxic stress has not been completely elucidated, but it could be associated with the concomitant activation of other pathways known to mediate survival, including NF-κB. In particular, Lamb et al. demonstrated that the AP-1 transcription factor JunD cooperates with NF-κB to increase the expression of prosurvival genes that contain both NF-κB- and AP-1-binding sites in their promoters. Because much of the cytotoxicity of chemotherapeutic agents occurs through apoptosis, the coactivation of NF-κB and AP-1, which can synergistically and effectively suppress the apoptotic potential of chemotherapeutic agents, could be a crucial obstacle to effective treatment of cancer
We recently demonstrated that an autocrine stimulation of interleukin 1 alpha (IL-1α), primarily mediated through induction of AP-1 activity, accounted for the constitutive activation of NF-κB and thus for the metastatic behavior of pancreatic cancer. During immune and inflammatory responses, detailed investigation of IL-1-induced tumor necrosis factor (TNF) receptor associated factor (TRAF)-6 signaling demonstrated activation of NF-κB through two parallel signaling pathways, depending on differential activation of two mitogen-activated protein kinase kinase kinases (MAP3Ks), MEKK3 (MAP3K3), or the TGF-β-activated kinase-1 (TAK1; MAP3K7).
TAK1 was originally identified as a MAP3K, which can be rapidly activated in response to TGF-β signal transduction. In vitro studies have demonstrated that overexpression of a dominant negative version of TAK1 inhibits both the activation of NF-κB and the mediator of AP-1 induction, c-Jun N-terminal kinase (JNK), thus increasing the sensitivity of cells to apoptosis induced by TNF-α. Mice carrying an epidermal-specific deletion of the TAK1 gene developed severe skin inflammation caused by impaired activation of NF-κB and JNK in response to TNF, which resulted in a massive apoptosis of keratinocytes much greater than those observed in IκB kinase beta (IKKβ) and IKKγ deletion models. A mouse model with TAK1 conditionally deleted in T cells was used to demonstrate that TAK1 is essential for in vivo thymocyte development and activation. The loss of TAK1 in the thymocytes prevented the activation of IKK, NF-κB, and JNK and sensitized the mutant cells to activation-induced apoptosis. Using a B cell-conditional TAK1-deficient mouse model, Sato et al. demonstrated that TAK1 is essential for toll-like receptor, IL-1 receptor, TNF receptor, and B cell receptor cellular responses and signaling pathways leading to the activation of JNK and/or NF-κB. Suppression of TAK1 signaling by dominant negative TAK1 reduced NF-κB activation in human head and neck squamous cell carcinoma and breast cancer cell lines.
Cellular inhibitor of apoptosis 2 (cIAP-2) is a member of the inhibitor of apoptosis (JAP) family of proteins that regulate programmed cell death by directly inhibiting caspases and by targeting proapoptotic components of the TNF-α signaling pathways for ubiquitin degradation. The overexpression of cIAP-2 is a common and early event in the progression of pancreatic cancer. Although the expression of cIAP-1 is constantly high in both normal and neoplastic pancreatic tissues, cIAP-2 mRNA levels are statistically significantly higher in pancreatic cancer than in normal pancreatic tissue. A sequence analysis of the cIAP-2 promoter revealed two critical NF-κB-binding sites and two potential AP-1-binding sites.
We hypothesized that TAK1 might be responsible for the resistance of pancreatic cancer to the proapoptotic effect of chemotherapeutic agents by increasing the NF-κB- and AP-1-mediated transcription of cIAP-2. Thus, targeting the expression or the kinase activity of TAK1 might reverse the intrinsic resistance of pancreatic cancer to chemotherapy.