Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer.

BackgroundUnderstanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFβ or activin-induced metastatic phenotype of colon cancer.MethodMitogenic signaling/growth factor receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (activin receptor 2) -/- or wild type mice. Colon cancer cell lines (+/- SMAD4) were interrogated for ligand-induced PI3K and MEK/ERK pathway activation and downstream protein/phospho-isoform expression/association after knockdown and pharmacologic inhibition of pathway members. EMT was assessed using epithelial/mesenchymal markers and migration assays.ResultsIn primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 expression was associated with TGFβ/MEK/ERK pathway activation. Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with activin-dependent PI3K signaling.ConclusionAlthough activin and TGFβ share growth suppressive SMAD signaling in colon cancer, they diverge in their SMAD4-independent pro-migratory signaling utilizing distinct mitogenic signaling pathways that affect EMT. p21 localization in colon cancer may determine a dominant activin versus TGFβ ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFβ family receptors

Simultaneous evolutionary expansion and constraint of genomic heterogeneity in multifocal lung cancer.

Recent genomic analyses have revealed substantial tumor heterogeneity across various cancers. However, it remains unclear whether and how genomic heterogeneity is constrained during tumor evolution. Here, we sequence a unique cohort of multiple synchronous lung cancers (MSLCs) to determine the relative diversity and uniformity of genetic drivers upon identical germline and environmental background. We find that each multicentric primary tumor harbors distinct oncogenic alterations, including novel mutations that are experimentally demonstrated to be functional and therapeutically targetable. However, functional studies show a strikingly constrained tumorigenic pathway underlying heterogeneous genetic variants. These results suggest that although the mutation-specific routes that cells take during oncogenesis are stochastic, genetic trajectories may be constrained by selection for functional convergence on key signaling pathways. Our findings highlight the robust evolutionary pressures that simultaneously shape the expansion and constraint of genomic diversity, a principle that holds important implications for understanding tumor evolution and optimizing therapeutic strategies.Across cancer types tumor heterogeneity has been observed, but how this relates to tumor evolution is unclear. Here, the authors sequence multiple synchronous lung cancers, highlighting the evolutionary pressures that simultaneously shape the expansion and constraint of genomic heterogeneity

ER stress in antigen‐presenting cells promotes NKT cell activation through endogenous neutral lipids

CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids

A Chemical-Genetic Screen for Identifying Substrates of the Er Kinase Perk

Cells constantly encounter changing environments that challenge the ability to adapt and survive. Signal transduction networks enable cells to appropriately sense and respond to these changes, and are often mediated through the activity of protein kinases. Protein kinases are a class of enzyme responsible for regulating a broad spectrum of cellular functions by transferring phosphate groups from ATP to substrate proteins, thereby altering substrate activity and function. PERK is a resident kinase of the endoplasmic reticulum, and is responsible for sensing perturbations in the protein folding capacity of the ER. When the influx of unfolded, nascent proteins exceeds the folding capacity of the ER, PERK initiates a cascade of signaling events that enable cell adaptation and ER stress resolution. These signaling pathways are not only essential for the survival of normal cells undergoing ER stress, but are also co-opted by tumor cells in order to survive the oxygen and nutrient-restricted conditions of the tumor microenvironment. Not surprisingly, PERK signaling is known to influence a variety of pro-tumorigenic processes. Therefore, from a purely biological standpoint as well as from a clinical perspective, it is important to understand this critical cell adaptive pathway in greater detail through identifying its interacting partners and thereby elucidating additional downstream signaling branches. Prior to the work described herein, only three direct PERK substrates had been identified. Using chemical-genetic screening techniques, we have generated a significant list of putative PERK substrates, several of which have been confirmed as PERK substrates in vitro. These preliminary results suggest new connections between known UPR pathways, as well as entirely novel signaling branches downstream of PERK. This work will provide a solid foundation for launching future PERK-related discovery studies

Protein Expression, Characterization and Activity Comparisons of Wild Type and Mutant DUSP5 Proteins

Background The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies

Targeting protein kinases to manage or prevent Alzheimer’s disease

Due to the pressing need for new disease-modifying drugs for Alzheimer’s disease (AD), new treatment strategies and alternative drug targets are currently being heavily researched. One such strategy is to modulate protein kinases such as cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 5 (CDK5), glycogen synthase kinase-3 (GSK-3α and GSK-3β), and the protein kinase RNA-like endoplasmic reticulum kinase (PERK). AD intervention by reduction of amyloid beta (Aβ) levels is also possible through development of protein kinase C-epsilon (PKC-ϵ) activators to recover α-secretase levels and decrease toxic Aβ levels, thereby restoring synaptogenesis and cognitive function. In this way, we aim to develop new AD drugs by targeting kinases that participate in AD pathophysiology. In our studies, comparative modeling was performed to construct 3D models for kinases whose crystal structures have not yet been identified. The information from structurally similar proteins was used to define the amino acid residues in the ATP binding site as well as other important sites and motifs. We searched for the comstructural motifs and domains of GSK-3β, CDK5 and PERK. Further, we identified the conserved water molecules in GSK-3β, CDK5 and PERK through calculation of the degree of water conservation. We investigated the protein-ligand interaction profiles of CDK1, CDK5, GSK-3α, GSK-3β and PERK based on molecular dynamics (MD) simulations, which provided a time-dependent demonstration of the interactions and contacts for each ligand. In addition, we explored the protein-protein interactions between CDK5 and p25. Small molecules which target this interaction may offer a prospective therapeutic benefit for AD. In order to identify new modulators for protein kinase targets in AD, we implemented three virtual screening protocols. The first protocol was a combined ligand- and protein structure-based approach to find new PERK inhibitors. In the second protocol, protein structure-based virtual screening was applied to find multiple-kinase inhibitors through parallel docking simulations into validated models of CDK1, CDK5 and GSK-3 kinases. In the third protocol, we searched for potential activators of PKC-ϵ based on the structure of its C1B domain

Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic–lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.Fil: Zhang, Lei. University Of South Dakota; Estados UnidosFil: Hapon, María Belén. University Of South Dakota; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Goyeneche, Alicia A.. University Of South Dakota; Estados Unidos. McGill University; CanadáFil: Srinivasan, Rekha. University Of South Dakota; Estados UnidosFil: Gamarra Luques, Carlos Diego. University Of South Dakota; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Callegari, Eduardo A.. University Of South Dakota; Estados UnidosFil: Drappeau, Donis D.. University Of South Dakota; Estados UnidosFil: Terpstra, Erin J.. University Of South Dakota; Estados UnidosFil: Pan, Bo. University Of South Dakota; Estados UnidosFil: Knapp, Jennifer R.. University of Kansas; Estados UnidosFil: Chien, Jeremy. University of Kansas; Estados UnidosFil: Wang, Xuejun. University Of South Dakota; Estados UnidosFil: Eyster, Kathleen M.. University Of South Dakota; Estados UnidosFil: Telleria, Carlos Marcelo. University Of South Dakota; Estados Unidos. McGill University; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Identification of novel modulators of protein synthesis and nucleolar biology using high throughput phenotypic screens

Protein synthesis and ribosome biogenesis are fundamental steps in gene expression and constitute the most energy demanding processes in living cells. Dysregulation of these processes is associated to a variety of human disorders including cancer, metabolic diseases, immunodeficiency, neurological and developmental disorders, and physiological aging. Therapeutic strategies modulating protein synthesis and ribosome biogenesis or nucleolar biology, have proven to be efficient for several of these disorders, and some of them are already used in the clinic, predominantly in the context of cancer. However, the success of these drugs has been limited due to activation of mechanisms of resistance or lack of general effects among different cancer types. Additionally, the application of modulators of protein and ribosome production in other disease contexts is just starting to be explored. This is particularly important for disorders where altered translation control is a hallmark, such as in the case of some neurodegenerative diseases. Moreover, different disorders may require different therapeutic approaches, hence, research in less known disease areas opens possibilities of finding new ways of regulating protein synthesis and ribosome biogenesis, and perhaps new biology. In this thesis we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology. Phenotypic screening allows for the systematic identification of regulators of an organismal feature (phenotype) without having any prior knowledge. In paper I we benefited from novel technologies allowing visualization of changes in protein synthesis to evaluate the effects of medically approved and well-characterized drugs in mRNA translation. Our screen failed to identify small molecules stimulating translation in cancer cells growing in complete media. Yet, it seems that translation can only be boosted when the translation machinery of cells is challenged, such as when cells are grown under starvation conditions. Nevertheless, our screen identified known down-regulators of translation, supporting the validity of our approach, and a new translation inhibitor, SKI-II. SKI-II was developed as a sphingosine kinase inhibitor (SPHK), and this group of compounds has been explored extensively as anticancer drugs. However, in our hands, SKI-II inhibited translation by inducing the integrated stress response (ISR), causing physical damage to the endoplasmic reticulum (ER), which resulted in cell death. The toxicity of SKI-II and its clinically relevant analog ABC294640 was not abrogated when knocking out sphingosine kinases, while it was partially rescued upon inhibition of the ISR. Our work is the first to systematically examine the effect of known drugs in translation in cells and to report cytotoxic properties of SPHK inhibitors that are independent of SPHKs. In paper II we conducted a chemical screen to identify compounds limiting the toxicity of amyotrophic lateral sclerosis (ALS)-related dipeptide repeats (DPRs). ALS is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, leading to muscular paralysis and death, within 3 to 5 years after diagnosis. The expansion of G4C2 repeats within the first intron of the C9ORF72 gene constitutes the most common cause of ALS and frontotemporal dementia (FTD). Through repeat-associated non-ATG (RAN) translation, these expansions are translated into DPRs, some of which, poly-proline arginine (PR) and poly-glycine arginine (GR), bind to the nucleoli and lead to cell death. Here we conducted a screen to identify compounds reducing toxicity of twenty-repeats poly-PR peptides (PR20) added exogenously to cells in culture. Our screen identified two BET bromodomain inhibitors (Bromosporine-1 and PFI-1) and sodium phenylbutyrate (Na-Phen), currently in clinical trials, as modifiers of PR20 toxicity in different cell lines and in developing zebrafish embryos. Our work shows that BET Bromodomain inhibitors rescue the nucleolar stress induced by PR20 and the known nucleolar stressor Actinomycin D (ActD). To our knowledge, this is the first time that compounds able to protect nucleolar integrity are reported in the literature, and therefore, they might have beneficial effects in diseases associated to nucleolar stress, such as ALS/FTD. Inspired by our results, we conducted four additional screens that are collected in the section preliminary results. Following paper I, we applied the same screening pipeline to identify novel modulators of translation among natural compounds (preliminary results I). Related to paper II, the literature points to two main issues with current modulators of ribosome biogenesis, promiscuity, even among the so-called selective modulators, and heterogeneity in the efficacy of compounds across different cancer types. Regarding the first, the discovery of regulators of ribosome biogenesis has advanced in parallel with the technology allowing their study. Current methods allow better characterization of the activities of these drugs and development of strategies to find more selective modulators, which we reviewed in annex I. Nevertheless, there is a growing need for novel modulators of nucleolar activity, and we benefited from publicly available image datasets to explore the effects of known drugs in the nucleolus (preliminary results II). Also, we conducted a genome-wide CRISPR/Cas9 screen to identify vulnerabilities to nucleolar stressors and systematically interrogate in which genetic backgrounds these drugs are suitable anticancer therapies (preliminary results III). Lastly, triggered by the discovery of “nucleolar protectors” in paper II, we conducted a chemical screen to explore novel nucleolar functions of known drugs using the Drug Repurposing Hub library 1 from the Broad Institute (preliminary results IV). Altogether, here we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology relevant for disease contexts, and to uncover new biology linked to these processes

Quantitative and systems pathology for therapeutic response prediction

The measurement of tissue biomarkers for therapeutic response prediction in cancer patients has become standard pathological practice, but only for a very limited number of targets. This is in spite of massive intellectual and financial investment in molecular pathology for translational cancer research. A re-evaluation of current approaches, and the testing of new ones, is required in order to meet the challenges of predicting responses to existing and novel therapeutics, and individualising therapy.Herein I critique the current state of tissue biomarker analysis and quantification in cancer pathology and the reasons why so few novel biomarkers have entered the clinic. In particular, we examine the central role of signalling pathway biology in sensitivity and resistance to targeted therapy. I discuss how accurate quantification, and the ability to simulate biological responses over time and space, may lead to more accurate prediction of therapeutic response. I propose that different mathematical techniques used in the nascent field of systems biology (ordinary differential equation-based, S-systems, and Bayesian approaches) may provide promising new avenues to improve prediction in clinical and pathological practice. I also discuss the challenges and opportunities for quantification in pathological research and practice.I have examined the role of cellular signalling pathways in therapeutic sensitivity and resistance in three different ways. Firstly, I have taken a hypothesis-driven and reductionist approach and shown that decreased Sprouty 2, a feedback inhibitor of MAPK and PI3K signalling, is associated with trastuzumab-resistance in vitro and in a cohort of breast cancer patients treated with trastuzumab. Secondly, I have characterised the activation state of ten growth and survival pathways across different histological subtypes of ovarian cancer using quantitative fluorescence microscopy. I have shown that unsupervised clustering of phosphoprotein expression profiles results in new subgroups with distinct biological properties (in terms of proliferation and apoptosis), and which predict therapeutic response to chemotherapy. Thirdly, I have developed a new mathematical model of PI3K signalling, parameterised using quantitative phosphoprotein expression data from cancer cell lines using reverse-phase protein microarrays, and shown that quantitative PTEN protein expression is the key determinant of resistance to anti-HER2 therapy in silico. Furthermore, the quantitative measurement of PTEN is more predictive of response than other pathway components taken in isolation and when tested by multivariate analysis in a cohort of breast cancers treated with trastuzumab. For the first time, a systems biology approach has successfully been used to stratify patients for personalised therapy in cancer, and is further compelling evidence that PTEN, appropriately measured in the clinical setting, refines clinical decision-making in patients treated with anti-HER2 therapies