Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most ...promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.
Despite intensive study, many mysteries remain about the MYCN oncogene's functions. Here we focus on MYCN's role in neuroblastoma, the most common extracranial childhood cancer. MYCN gene ...amplification occurs in 20% of cases, but other recurrent somatic mutations are rare. This scarcity of tractable targets has hampered efforts to develop new therapeutic options. We employed a multi-level omics approach to examine MYCN functioning and identify novel therapeutic targets for this largely un-druggable oncogene. We used systems medicine based computational network reconstruction and analysis to integrate a range of omic techniques: sequencing-based transcriptomics, genome-wide chromatin immunoprecipitation, siRNA screening and interaction proteomics, revealing that MYCN controls highly connected networks, with MYCN primarily supressing the activity of network components. MYCN's oncogenic functions are likely independent of its classical heterodimerisation partner, MAX. In particular, MYCN controls its own protein interaction network by transcriptionally regulating its binding partners.Our network-based approach identified vulnerable therapeutically targetable nodes that function as critical regulators or effectors of MYCN in neuroblastoma. These were validated by siRNA knockdown screens, functional studies and patient data. We identified β-estradiol and MAPK/ERK as having functional cross-talk with MYCN and being novel targetable vulnerabilities of MYCN-amplified neuroblastoma. These results reveal surprising differences between the functioning of endogenous, overexpressed and amplified MYCN, and rationalise how different MYCN dosages can orchestrate cell fate decisions and cancerous outcomes. Importantly, this work describes a systems-level approach to systematically uncovering network based vulnerabilities and therapeutic targets for multifactorial diseases by integrating disparate omic data types.
The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded ...genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.
Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the ...fusion oncogene
, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our
results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.
Abstract
The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional (2D) monolayers on plastic. However, many cellular features are impaired in these ...unnatural conditions and big alterations in gene expression in comparison to tumors have been reported. Three-dimensional (3D) cell culture models have become increasingly popular and are suggested to be better models than 2D monolayers due to improved cell-to-cell contacts and structures that resemble in vivo architecture.
The aim of this study was to develop a simple high-throughput 3D drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in 2D, in polyHEMA coated anchorage independent 3D models and in Matrigel on-top 3D cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating or they were allowed to grow in 3D for four days prior to the drug treatment. Big variations in drug responses were observed between the models indicating that comparisons of culture model influenced drug sensitivities cannot be made based on effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in 2D cultures, while responses of cells grown in polyHEMA resembled those of 2D. Furthermore, comparison of gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study we also suggest that 3D cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives for traditional 2D screens towards better comparability to in vivo state.
Citation Format: Vesa J. Hongisto, Sandra Nyberg, Vidal Fey, John-Patrick Mpindi, Olli Kallioniemi, Merja Perälä. High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells . abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3847. doi:10.1158/1538-7445.AM2013-3847
RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a ...RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets.
Abstract
Prostate cancer (PrCa) is the most common malignancy in Western countries and account for the second highest mortality rate of all cancer forms in males. Specifically in late stage ...castration-resistant PrCa (CRPC), the signaling pathways that contribute to invasion and metastasis are poorly understood. The heat shock factors (HSFs) are a family of multifaceted transcriptional regulators that govern survival upon proteotoxic stress. This is achieved, mainly via HSF1, through up-regulation of genes critical for endurance under stressful conditions. Interestingly, the capacity to withstand stress is also readily utilized by cancer cells, which face a variety of stressful conditions. It thus seems that HSF1 allows cancer cells to rapidly modify their physiology, metabolism and protein homeostasis, thereby facilitating oncogenesis. Moreover, lack of HSF1 reduces the susceptibility of mice to mutation- or carcinogen-driven tumors and reduces the growth of human cancer cell lines. Recently, examination of a cohort of breast cancer patients showed that high expression of HSF1 correlated with decreased survival rate, highlighting the clinical significance of HSF1.
The aim of this study was to elucidate novel molecular pathways that contribute to the detrimental development of CRPC. Our initial in silico analyses showed that in PrCa, HSF1 expression levels correlate with poor survival and high Gleason grades. Most intriguingly, HSF2, thus far unassociated with cancer, showed decreased expression in the same set of tumors. We used real time live cell imaging in combination with endpoint immunofluorescent stainings of 3-dimensional (3D) organotypic cultures of PC-3 cells that spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia and invasive structures. This model demonstrates the highly dynamic nature of epithelial plasticity, and the functional studies of 3D cultures implied roles for both of these factors in the invasive behavior of tumors. In fact, we found that HSF1 and HSF2 show opposing effect on tumor cell invasion. Tumor spheroids lacking HSF1 grew more slowly, were more differentiated and polarized, and were devoid of any invasive behavior. In contrast, downregulation of HSF2 seemed to increase the invasive properties, and HSF2 could therefore act as a tumor suppressor. In combination with bioinformatics analyses, these results were further validated in human pathology using tissue microarray and the molecular pathways behind these observations were elucidated by transcriptome analyses. Our results reveal involvement of two novel players in PrCa, HSF1 and HSF2 that intriguingly seem to have opposite roles in tumor invasion. We conclude that HSF1 and HSF2 have significant implications for the molecular understanding of cancer progression and invasion.
Citation Format: Malin Åkerfelt, Johanna Björk, Tuomas Mirtti, Vidal Fey, Lea Sistonen, Matthias Nees. Opposing effects of heat shock factors 1 and 2 on prostate cancer invasion. abstract. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A46.
Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor beta (TGF-beta) is released from bone during ...osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-beta induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-beta-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3' UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-beta signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chloroplasts are genetically semiautonomous organelles that contain their own subset of 100-120 genes coding for chloroplast proteins, tRNAs, and rRNAs. However, the great majority of the chloroplast ...proteins are encoded in the nucleus and must be imported into the organelle after their translation in the cytosol. This arrangement requires a high degree of coordination between the gene expression machineries in chloroplasts and nucleus, which is achieved by a permanent exchange of information between both compartments. The existence of such coordinating signals has long been known; however, the underlying molecular mechanisms and signaling routes are not understood. The present data indicate that the expression of nuclear-encoded chloroplast proteins is coupled to the functional state of the chloroplasts. Photosynthesis, which is the major function of chloroplasts, plays a crucial role in this context. Changes in the reduction/oxidation (redox) state of components of the photosynthetic machinery act as signals, which regulate the expression of chloroplast proteins in both chloroplasts and nucleus and help to coordinate the expression both in compartments. Recent advances in understanding chloroplast redox regulation of nuclear gene expression are summarized, and the importance for intracellular signaling is discussed.
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