Mitochondria : a target for anticancer therapy

Mitochondria possess a central role in several cellular metabolic pathways, maintenance of calcium homeostasis, production of reactive oxygen species (ROS) and in the regulation of various cell death modalities. A majority of cancers demonstrate aberrations in mitochondrial functions, which were shown to contribute to tumourigenesis. In addition, many mechanisms of chemotherapy-resistance are located upstream of the mitochondria in cell death pathways. Thus, destabilization of mitochondria and permeabilization of the outer mitochondrial membrane (OMM), a point of no return in apoptosis induction, represent promising strategies for anticancer therapy. One major aim of this thesis was to identify therapeutic approaches to overcome resistance of cancer cells to conventional chemotherapeutic drugs. We could show in Paper I that chemotherapy resistance mechanisms in cancers, mediated by various oncogenic signalling/mutations, could be overcome by targeting Complex II of the mitochondrial respiratory chain. Treatment of Neuroblastoma (NB) cells with α-tocopheryl succinate (α-TOS), a redox-silent analogue of vitamin-E, which was shown to target Complex II, mediate ROS-production and an increase of cytosolic calcium levels, could induce apoptosis in cancers cells irrespective of their MycN or p53 status. We propose that this is based on the ability of α-TOS to induce both mechanisms of OMM permeabilization, in a Bax/Bak-dependent manner, as well as calcium-dependent induction of mitochondrial permeability transition (MPT). In Paper II and III we investigated the possibility of sensitizing cancer cells to conventional anticancer drugs in a co-treatment setting with compounds targeting Complex II. In case of α-TOS (Paper II), the obtained results revealed contrasting effects for the chemotherapeutic drugs etoposide and cisplatin. In case of etoposide, α-TOS was able to sensitize cancer cells in a dose-dependent manner. Whereas strikingly, in case of cisplatin, low concentration of α-TOS protected cells from cisplatin-induced toxicity. We demonstrated that the succinate moiety of α-TOS is mediating this protective effect via stimulation of Complex II activity. However, when Complex II was inhibited using thenoyltrifluoroacetone (TTFA) (Paper III), a specific inhibitor of the ubiquinone binding site of Complex II, cells could be sensitized to both, etoposide- and cisplatin- induced cytotoxicity. This chemosensitizing effect was shown to rely on Complex II-mediated ROS-production. For the study that was concluded in Paper IV, a different approach was utilized. Citrate, a substrate of the tricarboxylic acid cycle, was shown to induce cytotoxicity in cells. The underlying mechanism was speculated to be based on citrate’s inhibitory effect on several crucial glycolytic enzymes and its ability to chelate calcium. We could demonstrate that although these features contribute, the main cause of cell death induced by citrate is the activation of initiator caspases. The underlying mechanism was proposed to be the kosmotropic property of citrate. In summary, the findings of this PhD thesis clearly underline the potency of exploiting mitochondria for anticancer therapy. Particularly Complex II plays an intriguing role in the sensitivity towards chemotherapy and represents an attractive target that should be further explored in future projects. In addition, new roles of well-known mitochondrial substrates were revealed

IKKβ kinase promotes stemness, migration, and invasion in KRAS-driven lung adenocarcinoma cells

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease

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 PGE 2 (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, PGE 2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner

Identification of a Clinically Relevant Signature for Early Progression in KRAS-Driven Lung Adenocarcinoma

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression

EC₅₀ of EP1-4 receptor antagonists on neuroblastoma cell viability <i>in vitro</i>.

<p>Abbreviations: EC₅₀; effective concentration decreasing neuroblastoma cell viability with 50%,</p>a<p>MYCN amplification;</p>b<p>Multidrug-resistant phenotype.</p

Neuroblastoma cells produce PGE₂ and dmPGE₂ increases cell viability.

<p>(A) Neuroblastoma cells produce PGE₂. SK-N-BE(2) and SK-N-SH cells were cultured with or without 40 µM of arachidonic acid (AA) for 48 h and 10 ng/mL IL-1β for 12 h. Cell homogenates were incubated with 80 µM of arachidonic acid and the concentration of produced PGE₂ was measured using LC-MS/MS. (B) PGE₂ increases neuroblastoma cell viability. SK-N-BE(2) and SK-N-SH cells were incubated in a serum-free medium for 24 h before adding different concentrations of dmPGE₂. Cell viability was measured using MTT-assay after 24, 48, 72 or 96 h. Values are representative of two independent experiments and data are expressed as mean (±SD) in percentage of control at 24 h. A statistical analysis was performed using 2-way ANOVA p<0.0001 for both concentration and incubation time. (C) PGE₂ rescues neuroblastoma cells from celecoxib induced apoptosis. SK-N-BE(2) cells were incubated in 35 µM celecoxib alone or in combination with 5 µM dmPGE₂. After 48 h cell viability was assessed using MTT-assay. Mean (±SD) of six replicate wells is shown; values are representative of three independent experiments. Statistical analysis was performed using 2-sided t test P<0.0001.</p

dmPGE₂ increases intracellular Ca²⁺ and cAMP concentrations followed by phosphorylation of Akt.

<p>(A) Intracellular calcium mobilization in response to dmPGE₂. SK-N-SH cells were loaded with the calcium fluorescent dye Fluo-4/AM before the addition of 1 µM dmPGE₂ or (B) pre-treatment with 2 mM EGTA before exposure to 1 µM dmPGE₂. The fluorescence intensity was followed using a confocal laser scanning microscope and representative single-cell recordings are shown. The arrows indicate when dmPGE₂ is added. (C) Intracellular accumulation of cAMP in response to dmPGE₂. SK-N-SH cells were incubated overnight in a medium without serum before the addition of 1 µM of dmPGE₂. Pretreatment with 10 µM NF 449, which is a Gαs inhibitor, before the incubation in dmPGE₂ for 10 min inhibited the production of cAMP. Forskolin, 10 µM for 10 min, was used as a positive control. The graph shows mean (±SD) in % of untreated control of three independent experiments. A statistical analysis was performed using 2-sided t-test, P<0.05. (D) PGE₂ induces phosphorlyation of Akt. SK-N-BE(2) and SK-N-SH cells were grown in the presence of serum (Ctr) before 24 h of culturing in the absence of serum (0 h) prior to the addition of 1 µM of dmPGE₂. Cells were further incubated in dmPGE₂ for 1, 2, 4, 6, 12 or 24 h and protein extracts were subjected to western blotting to detect phosphorylated Akt(ser473). An antibody detecting unphosphorylated Akt was used to exclude differences in total protein expression. β-actin was used to control for equal protein loading. The western blots are representative of three independent experiments.</p