Björn Kruspig

Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner. Conclusions These findings demonstrate that PGE2 acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE2 signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.

Citrate kills tumor cells through activation of apical caspases.

Most tumor cells exhibit a glycolytic phenotype. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Among the agents that can suppress glycolysis is citrate, a member of the Krebs cycle and an inhibitor of phosphofructokinase. Here, we show that citrate can trigger cell death in multiple cancer cell lines. The lethal effect of citrate was found to be related to the activation of apical caspases-8 and -2, rather than to the inhibition of cellular energy metabolism. Hence, increasing concentrations of citrate induced characteristic manifestations of apoptosis, such as caspase-3 activation, and poly-ADP-ribose polymerase cleavage, as well as the release of cytochrome c. Apoptosis induction did not involve the receptor-mediated pathway, since the processing of caspase-8 was not attenuated in cells deficient in Fas-associated protein with Death Domain. We propose that the activation of apical caspases by citrate could be explained by its kosmotropic properties. Caspase-8 is activated by proximity-induced dimerization, which might be facilitated by citrate through the stabilization of intermolecular interactions between the proteins.

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca2+ into the cytosol, compromised Ca2+ buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca2+ accumulation or chelation of cytosolic Ca2+ rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.

Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis

Targeting mitochondria is a promising strategy in tumor cell elimination. d-α-tocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, is a potentially powerful tool for fighting tumors by directly affecting mitochondria. However, when used at low concentrations it can suppress apoptosis induced by the conventionally used anticancer drug cisplatin. In cells treated with cisplatin, 30μM α-TOS prominently attenuated the manifestation of characteristic features of apoptosis - release of cytochrome c from mitochondria, caspase-3-like activity, and cleavage of poly(ADP-ribose) polymerase. In contrast, cell death induced by etoposide was not inhibited but rather stimulated by α-TOS. Thus, co-treatment with α-TOS and conventional antitumor drugs should be carried out with caution.

Mitochondrial substrates in cancer: Drivers or passengers?

The majority of cancers demonstrate various tumor-specific metabolic aberrations, such as increased glycolysis even under aerobic conditions (Warburg effect), whereas mitochondrial metabolic activity and their contribution to cellular energy production are restrained. One of the most important mechanisms for this metabolic switch is the alteration in the abundance, utilization, and localization of various mitochondrial substrates. Numerous lines of evidence connect disturbances in mitochondrial metabolic pathways with tumorigenesis and provide an intriguing rationale for utilizing mitochondria as targets for anti-cancer therapy.

CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1 , Pten and Nf1 , and correlation with platinum sensitivity

Transplantable murine models of ovarian high grade serous carcinoma (HGSC) remain an important research tool. We previously showed that ID8, a widely-used syngeneic model of ovarian cancer, lacked any of the frequent mutations in HGSC, and used CRISPR/Cas9 gene editing to generate derivatives with deletions in Trp53 and Brca2. Here we have used one ID8 Trp53−/− clone to generate further mutants, with additional mutations in Brca1, Pten and Nf1, all of which are frequently mutated or deleted in HGSC. We have also generated clones with triple deletions in Trp53, Brca2 and Pten. We show that ID8 Trp53−/−;Brca1−/− and Trp53−/−;Brca2−/− cells have defective homologous recombination and increased sensitivity to both platinum and PARP inhibitor chemotherapy compared to Trp53−/−. By contrast, loss of Pten or Nf1 increases growth rate in vivo, and reduces survival following cisplatin chemotherapy in vivo. Finally, we have also targeted Trp53 in cells isolated from a previous transgenic murine fallopian tube carcinoma model, and confirmed that loss of p53 expression in this second model accelerates intraperitoneal growth. Together, these CRISPR-generated models represent a new and simple tool to investigate the biology of HGSC, and the ID8 cell lines are freely available to researchers.

Contrasting effects of glutamine deprivation on apoptosis induced by conventionally used anticancer drugs

Tumor cells dependence on glutamine offers a rationale for their elimination via targeting of glutamine metabolism. The aim of this work was to investigate how glutamine deprivation affects the cellular response to conventionally used anticancer drugs. To answer this question, neuroblastoma cells were pre-incubated in a glutamine-free medium and treated with cisplatin or etoposide. Obtained results revealed that glutamine withdrawal affected cellular response to therapeutic drugs in a different manner. Glutamine deprivation suppressed etoposide-induced, but markedly stimulated cisplatin-induced apoptosis. Suppression of etoposide-induced cell death correlated with a downregulation of p53 expression, which, among other functions, regulates the expression of death receptor 5, one of the activators of caspase-8. In contrast, stimulation of cisplatin-induced cell death involved reactive oxygen species-mediated downregulation of FLIP-S, an inhibitor of caspase-8. As a result, the activity of caspase-8 was stimulated causing cleavage of the pro-apoptotic protein Bid, which is involved in the permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic factors, such as cytochrome c from mitochondria. Thus, suppression of glutamine metabolism can sensitize tumor cells to treatment and could be utilized for anti-cancer therapy. However, it should be done cautiously, since adverse effects may occur when combined with an inappropriate therapeutic drug.

The ERBB network facilitates KRAS-driven lung tumorigenesis

G12 mutant KRAS requires tonic ERBB network activity for initiation and maintenance of lung cancer. A new role for kinase inhibitors The KRAS oncogene is frequently mutated in a variety of cancer types, including lung cancer. Lung cancers with KRAS mutations are usually difficult to target, and conventional thinking dictates that these tumors are resistant to receptor tyrosine kinase inhibitors because those act upstream of the constitutively active KRAS protein. However, it appears that receptor tyrosine kinase signaling may have an effect on KRAS-driven lung tumors after all, by amplifying their growth beyond the effects of KRAS alone. Kruspig et al. and Moll et al. independently reached this conclusion and identified approved multi-kinase inhibitors that are effective in the setting of KRAS-mutant lung cancer in multiple mouse models, suggesting that this may be a potential treatment strategy for human patients as well. KRAS is the most frequently mutated driver oncogene in human adenocarcinoma of the lung. There are presently no clinically proven strategies for treatment of KRAS-driven lung cancer. Activating mutations in KRAS are thought to confer independence from upstream signaling; however, recent data suggest that this independence may not be absolute. We show that initiation and progression of KRAS-driven lung tumors require input from ERBB family receptor tyrosine kinases (RTKs): Multiple ERBB RTKs are expressed and active from the earliest stages of KRAS-driven lung tumor development, and treatment with a multi-ERBB inhibitor suppresses formation of KRASG12D-driven lung tumors. We present evidence that ERBB activity amplifies signaling through the core RAS pathway, supporting proliferation of KRAS-mutant tumor cells in culture and progression to invasive disease in vivo. Brief pharmacological inhibition of the ERBB network enhances the therapeutic benefit of MEK (mitogen-activated protein kinase kinase) inhibition in an autochthonous tumor setting. Our data suggest that lung cancer patients with KRAS-driven disease may benefit from inclusion of multi-ERBB inhibitors in rationally designed treatment strategies.

Targeting succinate:ubiquinone reductase potentiates the efficacy of anticancer therapy.

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1β inhibition with AKT activation in order to suppress GSK3β-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival.Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632-47. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.