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Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery:https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available athttps://phenaid.ardigen.com/jumpcpexplorer/.

The Hippo signaling cascade is a major pathway that integrates a broad spectrum of mechanosensory signals at the plasma membrane and regulates response via control of cell proliferation, self-renewal, differentiation, and apoptosis. Dysregulation of this pathway has been observed across a range of cancer types and results in an altered activity of its primary downstream effectors, the oncogenic transcription factors YAP/TAZ. For example, both germline and somatic loss-of-function mutations in the tumor suppressor gene NF2, a component of Hippo, induce hyperactivation of YAP/TAZ, transcriptional changes and ultimately result in tumor growth. The Hippo signaling pathway is an attractive target for drug discovery efforts, however, it is highly complex and still incompletely understood. Hence it is indispensable to get a deeper insight into the Hippo - YAP/TAZ signaling axis. To this end, we performed a genome-wide CRISPR knockout screen in the triple-negative breast cancer (TNBC) cell line MDA-MB231 (NF2LOF) expressing a YAP/TAZ reporter construct. We identified both negative and positive regulators of YAP/TAZ in breast cancer cells. In a second step, screening hits were further characterized in a focused single-cell CRISPR screen (Perturb-Seq), aiming at better understanding of the effects on YAP/TAZ activity regulation and downstream effects on gene expression. Here we present the technical details of our screening approaches and the results of perturbing known and novel regulators of YAP/TAZ on single cell level. We discuss the use of Perturb-Seq in the initial validation of hits from genome-wide screens and provide data that may serve as a basis for future drug discovery efforts, seeking for novel and effective treatments for triple-negative breast cancers and other malignancies with Hippo pathway alterations. Citation Format: Mareike Berlak, Zuzanna Makowska, Filippos Klironomos, Julia Kuehnlenz, Atanas Kamburov, Andreas Steffen, Martin Lange, Barbara Nicke, Ralf Lesche, Peter Staller, Charlotte Kopitz, Jan Naujoks. Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6089.

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYC induces massive lipid peroxidation upon depletion of cysteine, the rate-limiting amino acid for glutathione biosynthesis and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and irondependent type of cell death. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. MYCN may contribute to spontaneous tumor regression in low-risk neuroblastomas by promoting ferroptosis in cells with epigenetically silenced cystathionine-beta-synthase, the rate-limiting enzyme for transsulfuration. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.

Immune checkpoint blockade using antibodies targeting the cell surface expressed proteins CTLA-4, PD-1 and PD-L1 has revolutionized cancer care and its clinical impact in several indications has prompted a search for complementary immunostimulatory approaches that can further increase the efficacy of these drugs. Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1; HPK1), a serine/threonine kinase expressed exclusively in hematopoietic cell lineages, mediates a negative feedback signal downstream of T-cell receptor stimulation. Its activity is enhanced by PGE2 and TGFβ, factors commonly present in the tumor microenvironment. Mice deficient for MAP4K1 or expressing a kinase-dead variant of MAP4K1 exhibit enhanced T-cell function, including increased anti-tumor immunity. We developed the small molecule inhibitor BAY-405 that displays potent nanomolar MAP4K1 inhibition in biochemical and cellular assays, good kinase selectivity, and in vivo exposure after oral dosing. Pharmacological inhibition of MAP4K1 enhances T-cell immunity and overcomes the suppressive impact of PGE2, TGFβ and CD4+ T-regulatory cells. Single agent treatment of tumor-bearing mice results in suppression of tumor outgrowth in several syngeneic models. This is accompanied by an increase in the anti-tumor T-cell response, dependent on an intact T-cell compartment, while not involving direct anti-tumor cytotoxicity. Inhibition of MAP4K1 in conjunction with PD-L1 blockade results in further suppression of tumor outgrowth. Moreover, we found that MAP4K1 is expressed in both PD-L1-high and PD-L1-low human cancers. In summary, our data show that selective inhibition of MAP4K1 by means of small molecule drugs may be used to expand the patient population responding to immune checkpoint inhibition. Citation Format: Gabriele Leder, Rafael Carretero, Jeffrey Mowat, Sandra Berndt, Roland Neuhaus, Nuria Aiguabella Font, Ulf Boemer, Oliver von Ahsen, Uwe Eberspaecher, Judith Guenther, Mareike Grees, Corinna Link, Barbara Nicke, Daniel Baumann, Martina Schaefer, Mine Oezcan-Wahlbrink, Nicolas D. Werbeck, Ingo Hartung, Bertolt Kreft, Rienk Offringa. Enhancement of anti-tumor T-cell immunity by means of an oral small molecule targeting the intracellular immune checkpoint MAP4K1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1722.

Aberrant activation of the Hippo pathway effectors YAP1/TAZ promotes cell proliferation and tumorigenesis. To identify novel regulators of YAP1/TAZ in cancer, we established a FACS-based screening system monitoring YAP1/TAZ activity in MDA-MB-231 breast cancer cells. Using these cells, we performed pooled genome-wide CRISPR/Cas9 knockout and CRISPR activation/interference (a/i) screens. The list of hits included previously known YAP1/TAZ modulators such as LATS2, AJUBA, and TAZ itself, demonstrating the robustness of the screen. Moreover, we identified about 30 novel candidate genes with potential inhibitory activity on YAP1/TAZ and about 50 candidate genes that may play a role in YAP1/TAZ activation. These genes represent diverse cellular functions such as regulation of actin cytoskeleton, integrin signaling, and ER protein processing, among others. Modulation of endogenous YAP1/TAZ target genes was assessed by individual gene knockout using crRNAs. Functional characterization of the novel potential YAP1/TAZ modulators will aid to the further understanding of YAP1/TAZ biology in health and disease. Citation Format: Jan Naujoks, Lisette Potze, Julia Kuehnlenz, Atanas Kamburov, Ekaterina Nevedomskaya, Andreas Steffen, Claudia Luther, Anna Anurin, Anne Buttgereit, Stefan Prechtl, Benjamin Bader, Ralf Lesche, Peter Staller, Martin Lange, Barbara Nicke. Genome-wide CRISPR/Cas9 screens for the identification of novel YAP1/TAZ modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A29.

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.

Cancer stem cells (CSCs) are involved in metastasis and resistance development, thus affecting anticancer therapy efficacy. The underlying pathways required for CSC maintenance and survival are not fully understood and only a limited number of treatment strategies to specifically target CSCs have been identified. To identify novel CSC targeting compounds, we here set-up an aldehyde dehydrogenase (ALDH)-based phenotypic screening system that allows for an automated and standardized identification of CSCs. By staining cancer cells for ALDH activity and applying high-content-based single-cell population analysis, the proportion of a potential CSC subpopulation with significantly higher ALDH activity (ALDHhigh) can be quantified in a heterogeneous cell population. We confirmed high ALDH activity as surrogate marker for the CSC subpopulation in vitro and validated Wnt signaling as an essential factor for the maintenance of CSCs in SUM149 breast cancer cells. In a small molecule screen, we identified phosphodiesterase type 5 (PDE5) inhibition as potential strategy to target CSC maintenance and survival in multiple cancer cell lines. CSC elimination by PDE5 inhibition was not dependent on PKG signaling, and we suggest a novel mechanism in which PDE5 inhibition leads to elevated cGMP levels that stimulate cAMP/PKA signaling to eliminate CSCs.

Aberrant activation of the Hippo pathway effectors YAP1/TAZ promotes cell proliferation and tumorigenesis. To identify novel regulators of YAP1/TAZ as a possible means to treat cancer, we established a novel, FACS-based screening system monitoring YAP1/TAZ activity in MDA-MB-231 breast cancer cells. Using these cells, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. We identified approximately 50 genes potentially activating YAP1/TAZ with functions in the Actin Cytoskeleton signaling, p53 signaling, cell polarity or ER stress, amongst others. Moreover, we identified about 30 potential targets which when knocked out induce activity of YAP1/TAZ. The list of hits included genes known to affect the YAP1/TAZ activity such as AJUBA, LATS2 and TEAD, demonstrating the validity of the screen. Functional validation of the novel potential YAP1/TAZ modulators will aid to the further understanding of YAP1/TAZ biology and may open the door to new therapeutic avenues for targeting YAP1/TAZ in cancer. Citation Format: Jan Naujoks, Lisette Potze, Anna Anurin, Julia Kuehnlenz, Ralf Lesche, Atanas Kamburov, Ekaterina Nevedomskaya, Andreas Steffen, Martin Lange, Barbara Nicke. Genome-wide CRISPR/Cas9 screen for the identification of novel YAP1/TAZ modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3055.

Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT‐specific multidrug sensitization is associated with enhanced autophagosome–lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2+ breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2‐dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over‐expressed in HER2+ breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy‐treated patients with primary breast cancer. These data suggest that the induction of LAMP2‐dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra‐tumour heterogeneity. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. The purpose of the study was to analyze the interplay of oncogenic MYCN or c-MYC, referred to here as MYC(N), activity with cysteine metabolism and ferroptosis, an oxidative, non-apoptotic, and iron dependent form of regulated cell death caused by ROS-mediated massive lipid peroxidation (L-ROS), using MYC(N)-driven childhood neuroblastoma as a model.The intracellular amino acid levels at MYC(N)-high and MYC(N)-low cellular states were analyzed by HPLC. Effects on cell viability upon depletion of individual amino acids from the growth medium was tested in various cancer cell lines with regulable MYC(N). An unbiased high-throughput MYCN synthetic lethal siRNA screen was used to identify genes preferentially acting in the 'MYC(N)-high' state and protecting cells from ROS accumulation and ferroptosis. The capacity of cyst(e)ine uptake, intracellular cysteine synthesis via transsulfuration and glutathione biosynthesis was assessed in various neuroblastoma cell lines and tissues using metabolome, RNAseq, ChiP-seq and global proteome analyses. To investigate L-ROS formation at various conditions cells were stained with the lipid peroxidation sensor, C11-BODIPY, and flow cytometrically analyzed. Ferroptosis inducers (FINs) and inhibitors of transsulfuration were used to test their activity in various MYC(N)-dependent neuroblastoma cell lines and in vivo xenografts.We found that intracellular cysteine depletion in a 'MYC(N)-high' context induces cell death by ferroptosis and identified multiple points in glutathione synthesis and metabolism, particularly detoxification of L-ROS, that are vulnerable in the 'MYC(N)-high' state as compared to the 'MYC(N)-low' context. We could show that ferroptosis was dependent on MYC(N) expression and was enhanced by iron. We further demonstrated that both cystine import and intracellular cysteine synthesis via transulfuration achieved the intracellular state supportive of oncogenic MYC(N)-driven growth without endangering the cell to ferroptosis. We demonstrated the MYC(N) drives increased transsulfuration activity, rather than cysteine import, in tumor cells to maintain the cellular cysteine supply for glutathione synthesis. Our findings together with new descriptions of the ferroptotic process establish a novel functional link between oncogenic MYC(N) and ferroptosis, and imply regulation by cysteine-dependent glutathione availability. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically. Citation Format: Frank Westermann, Hamed Alborzinia, Sina Gogolin, Andrés F. Flórez, Lena M. Brückner, Moritz Gartlgruber, Sabine Hartlieb, Daniel Dreidax, Michal Nadler-Holly, Matthias Ziehm, Chunxuan Shao, Matthias Selbach, Carlo Stresemann, Gernot Poschet, Barbara Nicke, Stefan Wölfl, Kai O. Henrich, Thomas Höfer. MYCN mediates cysteine addiction and sensitizes to ferroptosis in cancer cells [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 4973.

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Targeted therapies in personalized medicine require the knowledge about the molecular changes within the patient that cause the disease. With the beginning of the new century, a plethora of new technologies became available to detect these changes and use this information as starting point for drug development. Next-generation genome sequencing and sophisticated genome-wide functional genomics' methods have led to a significant increase in the identification of novel drug target candidates and understanding of the relevance of these genomic and molecular changes for the diseases. As functional genomic tool for target identification, high-throughput gene silencing through RNA interference screening has become the established method. RNAi is discussed with its advantages and challenges in this chapter. Furthermore the potential of CRISPR/Cas9, a gene-editing method that has recently been adapted for use as functional screening tool, will be briefly reviewed.

Functional RNAi based screening is affected by large numbers of false positive and negative hits due to prevalent sequence based off-target effects. We performed a druggable genome targeting siRNA screen intended to identify novel regulators of E-cadherin (CDH1) expression, a known key player in epithelial mesenchymal transition (EMT). Analysis of primary screening results indicated a large number of false-positive hits. To address these crucial difficulties we developed an analysis method, SENSORS, which, similar to published methods, is a seed enrichment strategy for analyzing siRNA off-targets in RNAi screens. Using our approach, we were able to demonstrate that accounting for seed based off-target effects stratifies primary screening results and enables the discovery of additional screening hits. While traditional hit detection methods are prone to false positive results which are undetected, we were able to identify false positive hits robustly. Transcription factor MYBL1 was identified as a putative novel target required for CDH1 expression and verified experimentally. No siRNA pool targeting MYBL1 was present in the used siRNA library. Instead, MYBL1 was identified as a putative CDH1 regulating target solely based on the SENSORS off-target score, i.e. as a gene that is a cause for off-target effects down regulating E-cadherin expression.

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.

Targeted therapies in personalized medicine require the knowledge about the molecular changes within the patient that cause the disease. With the beginning of the new century, a plethora of new technologies became available to detect these changes and use this information as starting point for drug development. Next-generation genome sequencing and sophisticated genome-wide functional genomics' methods have led to a significant increase in the identification of novel drug target candidates and understanding of the relevance of these genomic and molecular changes for the diseases. As functional genomic tool for target identification, high-throughput gene silencing through RNA interference screening has become the established method. RNAi is discussed with its advantages and challenges in this chapter. Furthermore the potential of CRISPR/Cas9, a gene-editing method that has recently been adapted for use as functional screening tool, will be briefly reviewed.

Transforming growth factor-β (TGF-β) inhibits pancreatic acinar cell growth. In many cell types, TGF-β mediates its growth inhibitory effects by activation of Smad proteins. Recently, it has been reported that Smad proteins may interact with the mitogen-activated protein (MAP) kinase signaling pathways. In this study, we report on the interactions between the TGF-β and MAP kinase signaling pathways in isolated rat pancreatic acinar cells. TGF-β activated the MAP kinases extracellular signal-related kinases (ERKs) and p38 in pancreatic acinar cells, but had no effect on c- junNH2-terminal kinase activity. Activation of MAP kinase by TGF-β was maximal 4 h after treatment. The ability of TGF-β to activate ERKs was concentration dependent and dependent on protein synthesis. TGF-β's stimulation of ERK activation was blocked by PD-98059, an inhibitor of MAP kinase kinase 1, and by adenoviral transfer of dominant negative RasN17. Furthermore, adenoviral-mediated expression of dominant negative Smad4 blocked the ability of TGF-β to activate acinar cell MAP kinase, demonstrating that this activation is downstream of Smads. The biological relevance of ERK activation by TGF-β was indicated by demonstrating that inhibition of ERK signaling by PD-98059 blocked the ability of TGF-β to activate the transcription factor activator protein-1. These studies provide new insight into the signaling mechanisms by which TGF-β mediates biological actions in pancreatic acinar cells.

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these “CIN-survival” genes is associated with poor outcome in estrogen receptor–positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.

The sequential use of non cross-resistant cytotoxic agents is the standard of care for advanced solid tumors in order to enhance survival and optimise quality of life. Nevertheless, drug resistance to non cross-resistant agents is commonly witnessed, with clinical response rates to non cross-resistant regimens declining as the disease advances. Expression of ABC transporters is unlikely to fully explain this phenomenon, and a clear molecular explanation for this process remains uncertain. A statistical analysis of a recently published RNA interference screen targeting 779 kinases in three cell lines deriving from different tumor types reveals a significant correlation between resistance to paclitaxel and a non cross-resistant cytotoxic agent. Furthermore, 20% of kinases that promote resistance to paclitaxel when targeted by RNAi also promote resistance to a non cross-resistant agent within that same cell line, consistent with a tissue-type dependence of multi-drug resistance. Conversely, paclitaxel-specific resistance and sensitising kinases occur less frequently than expected. This indicates that several cell line specific kinases may regulate multi-drug resistance and provide a potential explanation for the phenomenon of drug resistance to non cross-resistant agents witnessed in oncology practice. Furthermore, this work supports efforts to identify common pathways of drug response for future drug discovery programmes.

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.

Publisher Summary This chapter focuses on the recombinant adenoviral expression of dominant-negative Ras N 17 blocking radiation-induced activation of mitogen-activated protein kinase pathway. Generation of recombinant adenoviruses to express a variety of proteins has become a method of choice for many investigators in the field of signal transduction. Using this technique, near 100% infection efficiencies can be obtained. A novel cellular target for ionizing radiation is shown to be the epidermal growth factor receptor (EGFR, also called ErbB 1), which is activated in response to irradiation of several carcinoma cell types. Radiation exposure, via the EGFR, can activate the extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK/MAPK) pathway to a level similar to that observed by physiologic EGF concentrations. The data presented in this chapter describe the generation of a recombinant adenovirus to express dominant-negative Ras N 17 and the effect this gene product has on radiation-induced ERK-MAPK activity.

Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.

Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.

In this study we used differential display reverse transcription-polymerase chain reaction to search for differentially expressed all-trans-retinoic acid (ATRA)-responsive genes in pancreatic carcinoma cells. We identified the kinesin-related protein HsEg5, which plays an essential role in spindle assembly and spindle function during mitosis, as a novel molecule involved in ATRA-mediated growth inhibition. Using Northern and Western blot analysis we demonstrated that ATRA significantly inhibits HsEg5 expression in various pancreatic carcinoma cell lines as well as in HaCat keratinocytes. Inhibition of HsEg5 expression by ATRA occurs at the posttranscriptional level. As a consequence, tumor cells synchronized in S-phase revealed a retarded progression through G2/M phase of the cell cycle indicating that HsEg5 inhibition results in a delayed progression through mitosis. Furthermore, a significant decrease of HsEg5 protein expression achieved by antisense transfection revealed a significant growth inhibition compared with control cells. Therefore, HsEg5 represents a novel molecule involved in ATRA-mediated growth inhibition, suggesting that vitamin A derivatives can interact with the bipolar spindle apparatus during mitosis.

CCK stimulates pleiotrophic responses in pancreatic acinar cells; however, the intracellular signaling pathways involved are not well understood. To evaluate the role of the ras gene product in CCK actions, a strategy involving in vitro adenoviral-mediated gene delivery of a dominant-negative mutant Ras (RasN17) was utilized. Isolated acini were infected with various titers of either a control adenovirus or an adenoviral construct expressing RasN17 for 24 h before being treated with CCK. Titer-dependent expression of RasN17 in the acini was confirmed by Western blotting. Infection with control adenovirus [106–109plaque-forming units/mg acinar protein (multiplicity of infection of ∼1–1,000)] had no effect on CCK stimulation of acinar cell amylase release, extracellular-regulated kinase (ERK) or c-Jun kinase (JNK) kinases, or DNA synthesis. In contrast, infection with adenovirus bearing ras N17 increased basal amylase release, inhibited CCK-mediated JNK activation, had no effect on CCK activation of ERK, and inhibited DNA synthesis. These data demonstrate important roles for Ras in specific actions of CCK on pancreatic acinar function.

The European Union multi-disciplinary Personalised RNA interference to Enhance the Delivery of Individualised Cytotoxic and Targeted therapeutics (PREDICT) consortium has recently initiated a framework to accelerate the development of predictive biomarkers of individual patient response to anti-cancer agents. The consortium focuses on the identification of reliable predictive biomarkers to approved agents with anti-angiogenic activity for which no reliable predictive biomarkers exist: sunitinib, a multi-targeted tyrosine kinase inhibitor and everolimus, a mammalian target of rapamycin (mTOR) pathway inhibitor. Through the analysis of tumor tissue derived from pre-operative renal cell carcinoma (RCC) clinical trials, the PREDICT consortium will use established and novel methods to integrate comprehensive tumor-derived genomic data with personalized tumor-derived small hairpin RNA and high-throughput small interfering RNA screens to identify and validate functionally important genomic or transcriptomic predictive biomarkers of individual drug response in patients. PREDICT's approach to predictive biomarker discovery differs from conventional associative learning approaches, which can be susceptible to the detection of chance associations that lead to overestimation of true clinical accuracy. These methods will identify molecular pathways important for survival and growth of RCC cells and particular targets suitable for therapeutic development. Importantly, our results may enable individualized treatment of RCC, reducing ineffective therapy in drug-resistant disease, leading to improved quality of life and higher cost efficiency, which in turn should broaden patient access to beneficial therapeutics, thereby enhancing clinical outcome and cancer survival. The consortium will also establish and consolidate a European network providing the technological and clinical platform for large-scale functional genomic biomarker discovery. Here we review our current understanding of molecular mechanisms driving resistance to anti-angiogenesis agents, the current limitations of laboratory and clinical trial strategies and how the PREDICT consortium will endeavor to identify a new generation of predictive biomarkers.

BACKGROUND—The molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood. AIM—To identify the intracellular mechanisms responsible for resistance against retinoid mediated growth inhibition in human colon carcinoma cells. METHODS—Anchorage independent growth of the human colon carcinoma cell lines HT29 and LoVo was determined by a human tumour clonogenic assay. Retinoid receptor expression was evaluated by reverse transcription polymerase chain reaction and northern blotting. Retinoid mediated transactivation was assessed by transient transfection of a pTK::βREx2-luc reporter construct. Retinoid receptor overexpression was achieved by selecting stably transfected cell clones. RESULTS—Retinoid treatment resulted in profound dose dependent growth inhibition in HT29 cells, while LoVo cells were unaffected. The two cell lines express identical patterns of nuclear retinoid receptor mRNA transcripts. However, on retinoid treatment, retinoic acid receptor β gene expression was upregulated only in retinoid sensitive HT29 cells, but not in retinoid resistant LoVo cells. In accordance, stable overexpression of retinoic acid receptor β but not α or γ conferred retinoid mediated growth inhibition on LoVo cells. CONCLUSION—Induction of retinoic acid receptor β expression is required and sufficent to confer retinoid mediated growth inhibition on human colon carcinoma cells. Keywords: colon cancer; retinoic acid; retinoic acid receptor

SummaryCytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.