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BackgroundTherapeutic management of stage II colon cancer remains difficult regarding the decision whether adjuvant chemotherapy should be administered or not. Low rates of recurrence are opposed to chemotherapy induced toxicity and current clinical features are limited in predicting disease relapse. Predictive biomarkers are urgently needed and we hypothesise that the spatial tissue composition of relapsed and non-relapsed colon cancer stage II patients reveals relevant biomarkers.MethodsThe spatial tissue composition of stage II colon cancer patients was examined by in situ sequencing technology with sub-cellular resolution. A panel of 175 genes was designed investigating specific cancer-associated processes and components of the tumour microenvironment. We identified a tumour gene signature to subclassify tissue into neoplastic and non-neoplastic tissue compartments based on spatial expression patterns generated by in situ sequencing (GTC-tool – Genes-To-Count).ResultsThe GTC-tool automatically identified tissue compartments that were used to quantify gene expression of biological processes upregulated within the neoplastic tissue in comparison to non-neoplastic tissue and within relapsed versus non-relapsed stage II colon patients. Three differentially expressed genes (FGFR2, MMP11 and OTOP2) in the neoplastic tissue compartments of relapsed patients in comparison to non-relapsed patients were identified predicting recurrence in stage II colon cancer.ConclusionsIn depth spatial in situ sequencing revealed novel potential predictive biomarkers for disease relapse in colon cancer stage II patients. Our developed open-access GTC-tool allows to accurately capture the tumour compartment and quantify spatial gene expression in colon cancer tissue.

We recently identified pathogenic KIF1Bβ mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bβ in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bβ is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bβ mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bβ-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bβ. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bβ independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bβ loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bβ mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.

KIF1Bβ is a candidate 1p36 tumor suppressor that regulates apoptosis in the developing sympathetic nervous system. We found that KIF1Bβ activates the Ca(2+)-dependent phosphatase calcineurin (CN) by stabilizing the CN-calmodulin complex, relieving enzymatic autoinhibition and enabling CN substrate recognition. CN is the key mediator of cellular responses to Ca(2+) signals and its deregulation is implicated in cancer, cardiac, neurodegenerative, and immune disease. We show that KIF1Bβ affects mitochondrial dynamics through CN-dependent dephosphorylation of Dynamin-related protein 1 (DRP1), causing mitochondrial fission and apoptosis. Furthermore, KIF1Bβ actuates recognition of all known CN substrates, implying a general mechanism for KIF1Bβ in Ca(2+) signaling and how Ca(2+)-dependent signaling is executed by CN. Pathogenic KIF1Bβ mutations previously identified in neuroblastomas and pheochromocytomas all fail to activate CN or stimulate DRP1 dephosphorylation. Importantly, KIF1Bβ and DRP1 are silenced in 1p36 hemizygous-deleted neuroblastomas, indicating that deregulation of calcineurin and mitochondrial dynamics contributes to high-risk and poor-prognosis neuroblastoma.

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

The consequences of DNA damage depend on the cell type and the severity of the damage. Mild DNA damage can be repaired with or without cell-cycle arrest. More severe and irreparable DNA injury leads to the appearance of cells that carry mutations or causes a shift towards induction of the senescence or cell death programs. Although for many years it was argued that DNA damage kills cells via apoptosis or necrosis, technical and methodological progress during the last few years has helped to reveal that this injury might also activate death by autophagy or mitotic catastrophe, which may then be followed by apoptosis or necrosis. The molecular basis underlying the decision-making process is currently the subject of intense investigation. Here, we review current knowledge about the response to DNA damage and subsequent signaling, with particular attention to cell death induction and the molecular switches between different cell death modalities following damage.

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development. We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug action can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues. Although CETSA was first developed for evaluating drug binding to target proteins in cells it is also useful for characterizing physiological interactions, including protein-protein, protein-metabolite and protein-nucleic acid interactions (Martinez Molina & Nordlund, Ann Rev Pharm Toxic 2016 56:141). In the proteome-wide CETSA experiment, interactions with more than 7000 proteins can be measured in parallel (Savitski, Science 2014 346;6205). This strategy therefore constitutes a novel mean for discovering key interactions determining the fate of cancer cells during therapy and resistance – interactions that can serve as biomarkers or candidates for novel drug targets in cancer therapy. Citation Format: Ying Yu Liang, Anderson Ramos, Henriette Laursen, Olga Surova, Johan Lengqvist, Sara Lööf, Anette Langebäck, Smaranda Bacanu, Jonas Bergh, Pär Nordlund. Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5655.

// Anna Zagryazhskaya 1 , Olga Surova 1, 2 , Nadeem S. Akbar 1 , Giulia Allavena 1, 3 , Katarina Gyuraszova 1, 4 , Irina B. Zborovskaya 5, 6 , Elena M. Tchevkina 5, 6 , Boris Zhivotovsky 1, 6 1 Institute of Environmental Medicine, Division of Toxicology, Stockholm, Sweden 2 Ludwig Institute for Cancer Research Ltd, Karolinska Institutet, Stockholm, Sweden 3 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 4 Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Safarik University in Kosice, Kosice, Slovakia 5 NN Blokhin Russian Cancer Research Center, Moscow, Russia 6 Faculty of Fundamental Medicine, ML Lomonosov State University, Moscow, Russia Correspondence to: Boris Zhivotovsky, e-mail: Boris.Zhivotovsky@ki.se Keywords: tudor staphylococcal nuclease, S100A11, phospholipase A 2 , non-small cell lung cancer, apoptosis Received: December 19, 2014 Accepted: March 07, 2015 Published: March 26, 2015 ABSTRACT Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF 3 , a phospholipase A 2 (PLA 2 ) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA 2 inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF 3 , as well as N -acetyl- L -cysteine, which also mimicked the effect of PLA 2 inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA 2 axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.

The consequences of DNA damage depend on the cell type and the severity of damage. Mild DNA damage can be repaired with or without cell cycle arrest. More severe and irreparable DNA damage leads to the appearance of cells that carry mutations, or causes a shift towards induction of the senescence or cell death programme. Although for many years it was argued that DNA damage kills cells via apoptosis or necrosis, technical and methodological progress during the last few years has helped reveal that DNA damage might also activate death by autophagy or mitotic catastrophe, which may then be followed by apoptosis or necrosis. The molecular basis underlying the decision-making process is currently the subject of intense investigation. Here we review current knowledge about the response to DNA damage and subsequent signalling, with particular attention to cell death induction and the molecular switches between different cell death modalities following damage.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising drug for the treatment of tumors; however, a number of cancer cells are resistant to this cytokine. Among the mechanisms of resistance of small cell lung carcinomas (SCLCs) to TRAIL is the lack of caspase-8 expression. Although methylation of the caspase-8 promoter has been suggested as the main mechanism of caspase-8 silencing, we showed that reduction of the enzymes involved in DNA methylation, DNA methyltransferases (DNMT) 1, 3a and 3b, was not sufficient to significantly restore caspase-8 expression in SCLC cells, signifying that other mechanisms are involved in caspase-8 silencing. We found that combination of the DNMT inhibitor decitabine with an inhibitor of histone deacetylase (HDAC) significantly increased caspase-8 expression in SCLC cells at the RNA and protein levels. Among all studied HDAC inhibitors, valproic acid (VPA) and CI-994 showed prolonged effects on histone acetylation, while combination with decitabine produced the most prominent effects on caspase-8 re-expression. Moreover, a significant reduction of survivin and cIAP-1 proteins level was observed after treatment with VPA. The combination of two drugs sensitized SCLC cells to TRAIL-induced apoptosis, involving mitochondrial apoptotic pathway and was accompanied by Bid cleavage, activation of Bax, and release of cytochrome c. Both initiator caspase-8 and -9 were required for the sensitization of SCLC cells to TRAIL. Thus, efficient restoration of caspase-8 expression in SCLC cells is achieved when a combination of DNMT and HDAC inhibitors is used, suggesting a combination of decitabine and VPA or CI-994 as a potential treatment for sensitization of SCLC cells lacking caspase-8 to TRAIL.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIPS) was significantly increased in surviving cells. Such upregulation of c-FLIPS was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIPS, but not c-FLIP-long (c-FLIPL), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca(2+) or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIPS, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIPS, and it is suggested that blocking c-FLIPS expression by inhibition of Ca(2+)/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.

Lung cancer (LC) is among the most common tumors liable for worldwide highest mortality rate. It has a poor prognosis and symptoms related to disease occur at an advanced stage where therapy response becomes less effective. Resistance to radio- and chemotherapy of all tumors in general and LC, in particular, is a complex phenomenon. Evasion of cell death is one of the key hallmarks of both tumorigenesis and resistance to treatment. Differential expression of microRNAs (miRNAs or miRs), a class of small non-coding RNAs, 19-25 nucleotides in length, and proteins involved in miRNAs maturation (Drosha, Exportin-5, Dicer1, PACT, Tudor-SN, Argonaute-2, and FXR1) have been linked to carcinogenesis and therapy resistance. Nevertheless, comprehensive information is needed to better understand their function in tumor formation and resistance/sensitization to therapy. Therefore, the aim of this study was to investigate the role of miRNA machinery in chemoresistance of a selected panel of non-small cell lung carcinoma (NSCLC) cell lines (U1810, H661 and A549) resistant to treatment. It was revealed that knockdown of Drosha, Dicer-1 and Ago-2 was not sufficient to sensitize them to subsequent treatment with either cisplatin, 5-FU, etoposide or camptothecin (CPT), while silencing of Staphylococcal nuclease domain containing 1(SND1/p100/TSN), a key component of RISC complex, led to increased sensitivity of NSCLC cells to the DNA-damaging agents. Besides its function as a component of the multiprotein complex involved in miRNA functioning, TSN is known to act as a transcriptional activator and oncogene in many cancers. Additionally, it is cleaved during apoptosis, losing its pro-carcinogenic function. Our results show that these NSCLC cells with TSN-knocked down exhibited much stronger response to treatment with CPT and cisplatin compared to wild-type cells, as monitored by enhanced cleavage of PARP, increase in percentage of subG1 cells and potentiation of caspase-3-like activity. Overall, obtained data suggest that this nuclease can play an important role in the resistance of NSCLC cells to chemotherapy and can be used as a potential target to increase sensitivity of lung tumors to this type of treatment. 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 4882. doi:1538-7445.AM2012-4882