Cancer Cell Drug Response Research Articles

Controlled drug release with surface-capped mesoporous silica nanoparticles and its label-free in situ Raman monitoring

Mesoporous silica nanoparticles (MSNs) have drawn attention as efficient nanocarriers for drug delivery systems owing to their unique physiochemical properties. However, systemically controlling the kinetics of drug release from the nanocarriers and in situ monitoring of the drug release are still challenging. Here, we report surface-capped MSNs used for controlled drug release and demonstrate label-free in situ Raman monitoring of released drugs based on the molecule-specific spectral fingerprints. By capping the surface of MSNs with amine moieties, gold nanoparticles, and albumin, we achieved high loading efficiencies (up to 97%) of doxorubicin and precisely controlled drug release stimulated by changing pH value. Moreover, we monitored in real-time drug release profile and visualized cellular distribution of the delivered drug at nanoscale based on its intrinsic Raman peak. Finally, we evaluated drug responses in cancer cells and normal cells to investigate whether capped-dMSNs exhibit selective drug release. Our findings would be beneficial for designing smart drug carriers and directly monitoring the release behavior of drugs in actual cellular environments.

Identification of Cdk1-LATS-Pin1 as a Novel Signaling Axis in Anti-tubulin Drug Response of Cancer Cells.

The Hippo pathway is a signaling cascade that plays important roles in organ size control, tumorigenesis, metastasis, stress response, stem cell differentiation, and renewal during development and tissue homeostasis and mechanotransduction. Recently, it has been observed that loss of the Hippo pathway core component LATS (large tumor suppressor) or overexpression of its downstream targets YAP and its paralog TAZ causes resistance of cancer cells to anti-tubulin drugs. However, YAP and TAZ mediates anti-tubulin drug-induced apoptosis independent of its upstream regulator LATS and the Hippo pathway. Thus, the underlying molecular mechanism of how LATS is involved in the anti-tubulin drug response remains unknown. Proteomic approaches, SILAC and BioID, were used to identify the isomerase Pin1 as a novel LATS-interacting protein after anti-tubulin drug treatment. Treatment with anti-tubulin drugs activated cyclin-dependent kinase 1 (CDK1), which phosphorylates LATS2 at five S/T-P motifs that functionally interact with the WW domain of Pin1 and inhibit its antiapoptotic function. Thus, these data identify Cdk1 and Pin1 as a novel upstream regulator and downstream mediator, respectively, of LATS in antitubulin drug response. Further studies on this novel Cdk1-LATS-Pin1 signaling axis will be important for understanding the molecular mechanisms of drug resistance and will provide useful information for targeting of this pathway in the future.Implications: This study provides new insight on the molecular mechanism of anti-tubulin drug resistance and suggests novel therapeutic targets for drug-resistant cancers. Mol Cancer Res; 16(6); 1035-45. ©2018 AACR.

Abstract 5560: Systemic map of protein phosphatase 2A (PP2A)-regulated phosphotargets and drug responses in cancer cells

Abstract Despite the pivotal role of phosphatases in cancer cell signalling, systemic understanding of phosphatase targets is still at infancy. Protein phosphatase 2A (PP2A) is a human tumor suppressor complex. PP2A inhibition is a requirement for human cell transformation and PP2A regulates many cancer critical signalling pathways. Importantly, emerging data indicates that reactivation of PP2A tumor suppressor activity could provide entirely novel approach for cancer therapy. Here, we present first systemic analysis of phosphoprotein targets (dephosphorylome) regulated by PP2A in cancer cells. Based on data, PP2A regulates cancer critical signalling pathways, including entire EGFR-RAS-RAF-MEK-ERK cascade, and functions as a master regulator of MYC function. At network level, PP2A targets critical cellular processes such as chromosome organization, RNA splicing, and nuclear envelope assembly. Surprisingly, soft clustering of PP2A dephosphorylome revealed that most phospho-target residues are subject to only unidirectional regulation in cancer cells. Moreover, targets show intracellular gradient where phosphatase inhibition dominates nuclear phosphorylation balance. Since phosphoregulation is critical for cancer drug responses, dephosphorylome was correlated with cancer cell responses to over 300 drugs. Importantly, cancer therapies could be broadly classified based on their dephosphorylome, both at quantitative and qualitative manner. Finally, we demonstrate the utility of this large dataset by validating the role of PP2A in MEK inhibitor resistance in KRAS mutant cancer cells via regulation of RAF, mTor and MYC. The study presents first systemic resource to understand potential of PP2A manipulation in cancer cell signalling and drug responses. The study also uncovers generally important insights to phosphoregulation. Citation Format: Otto Kauko, Susumu Imanishi, Evgeny Kulesskiy, Teemu D. Laajala, Laxmana Yetukuri, Artur Padzik, Mikael Jumppanen, Pekka Haapaniemi, Bhagwan Yadaw, Veronika Suni, Taru Varila, Garry Corthals, Wennerberg Krister, Tero Aittokallio, Jukka Westermarck. Systemic map of protein phosphatase 2A (PP2A)-regulated phosphotargets and drug responses in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2017-5560

Inferences of drug responses in cancer cells from cancer genomic features and compound chemical and therapeutic properties.

Accurately predicting the response of a cancer patient to a therapeutic agent is a core goal of precision medicine. Existing approaches were mainly relied primarily on genomic alterations in cancer cells that have been treated with different drugs. Here we focus on predicting drug response based on integration of the heterogeneously pharmacogenomics data from both cell and drug sides. Through a systematical approach, named as PDRCC (Predict Drug Response in Cancer Cells), the cancer genomic alterations and compound chemical and therapeutic properties were incorporated to determine the chemotherapeutic response in cancer patients. Using the Cancer Cell Line Encyclopedia (CCLE) study as the benchmark dataset, all pharmacogenomics data exhibited their roles in inferring the relationships between cancer cells and drugs. When integrating both genomic resources and compound information, the prediction coverage was significantly increased. The validity of PDRCC was also supported by its effective in uncovering the unknown cell-drug associations with database and literature evidences. It set the stage for clinical testing of novel therapeutic strategies, such as the sensitive association between cancer cell ‘A549_LUNG’ and compound ‘Topotecan’. In conclusion, PDRCC offers the possibility for faster, safer, and cheaper the development of novel anti-cancer therapeutics in the early-stage clinical trails.

Computational imaging reveals mitochondrial morphology as a biomarker of cancer phenotype and drug response.

Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations’ morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.

Abstract 2791: Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines

Abstract Background Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients’ response to therapy. The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including acute myeloid leukemia (AML), Ewings sarcoma (ES), osteosarcoma (OS), and neuroblastoma (NB), under normoxia and hypoxia, and (2) to determine the potential mechanisms of drug resistance under hypoxia. Methods and Results In the present study the IC50 values were determined for the following targeting agents: linsitinib, picropodophylin (insulin-like growth factor-1 receptor), YM155 (survivin), AZD 8055 (mTOR), dovitinib (FLT3, c-kit, FGF4), and Palbociclib (CDK4/CDK6) in a panel of 15 AML, 4 ES, 2 OS and 2 NB cell lines using Cell Titer Glo assay. The effect of hypoxia was differential. It depended on the cell line and the compound used. However, hypoxia induced universal resistance to YM155 in all cell lines tested. YM155 had significant cytotoxicity in AML cell lines (IC50 between 2.5-1775 nM) in normoxia, however, the IC50 values in hypoxia were in the micromolar range, with an increase of > 100 fold. The effect of hypoxia on YM155 response in ES cell lines was less pronounced with (IC50s between 1-10 nM) in normoxia compared to (IC50 between 6 and 350 nM) in hypoxia. We studied the effect of hypoxia on the cell cycle using propidium iodide and flow cytometry in MV4-11 AML cells. Treatment with YM155 for 24 h in normoxia induced a pronounced decrease in the percentage of cells in S phase and G2/M phase and an increase in the sub-G1 phase. However, no change in cell cycle phases was detected under hypoxia. Ongoing studies are focusing on determining the mechanisms of hypoxia-induced YM155 resistance through studying the expression of proteins regulating hypoxia, cell cycle, and apoptosis using western blot. Conclusion These data demonstrate that hypoxia induced different effects in a panel of pediatric cancer cell lines. However, it conferred pronounced resistance to YM155 in AML and ES cell lines. Hypoxia is likely a key factor in programming treatment resistance and, thus, relapse and poor outcomes. Citation Format: Justin Montoya, Kieran Finch, Jan Simper, Robert J. Arceci, Eiman A. Aleem. Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2791.

Abstract 234: Mitochondrial morphology as a biomarker of cancer phenotype and drug response

Abstract Mitochondria, critical organelles in quiescent and dividing cells, have been shown to vary in number, mass, and shape. While previous work has focused on understanding molecular mechanisms underlying changes in normal and immortalized cells, less is known about longer-term mitochondrial behavior such as morphological patterns in individual cells and overall populations, the heterogeneity and functional consequences of these patterns, and ultimately how potential mitochondrial heterogeneity can be exploited in the clinic. Indeed, while previous studies have been conducted illustrating mitochondrial dysfunction and morphological alterations in certain tumor types, more comprehensive analyses across tumor and cell types, as well as from cells derived from individual patients, have yet to be conducted. A primary impediment to this work is the technical challenge of consistently assaying mitochondrial morphology in diverse cell types where fixation procedures, collection techniques, and differing optical qualities between samples make potential analyses a challenging application. We have developed an image based analytical technique to phenotype mitochondrial morphology in different cancers, including in vitro cancer cell lines and patient derived cancer cells. Our methodology is based on a combination of novel, clinically relevant collection and fixation techniques, and advanced image processing and computer learning analysis methodologies. Our results demonstrate that i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; ii) a given phenotype is characteristic of a cell population and fairly constant over time; iii) mitochondrial patterns correlate with cell metabolomics measurements and iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. Citation Format: Randy J. Giedt, Paolo Fumene Fergulio, Divya Pathania, Katherine S. Yang, Aoife Kilcoyne, Claudio Vinegoni, Tim J. Mitchison, Ralph Weissleder. Mitochondrial morphology as a biomarker of cancer phenotype and drug response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 234.

Cancer stem cell drugs target K-ras signaling in a stemness context.

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.

Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells' sensitivity to paclitaxel.

The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3′ UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy.

Abstract A61: Tumor metabolic remodeling can modulate anticancer drug response: CPI-613 attack on tumor cell mitochondrial metabolism is mediated by metabolite availability

Abstract We will describe recent progress in understanding the role of cancer metabolic remodeling in determining the response of tumors and tumor cells to a novel, first-in-class clinical agent class selectively targeting cancer cell mitochondrial metabolism. The reconfiguration of mitochondrial metabolism in cancer in general, and the tricarboxylic acid (TCA) cycle in particular, presents a rich source of potential therapeutic targets. Pyruvate dehydrogenase (PDH) and alpha ketoglutarate dehydrogenase (KGDH) are two gatekeeper enzymes regulating carbon flux into the mitochondrial TCA cycle. A key component of the altered regulatory processes in cancer is lipoic acid (lipoate), which not only functions as a cofactor for both PDH and KGDH, but also as source of regulatory information. Moreover, the lipoate-sensitive regulatory components of PDH and KGDH are substantially reconfigured in tumor cells, providing cancer-specific targets that can be successfully attacked with properly designed lipoate analogs. We developed and characterized CPI-613, a lipoic acid analog, which selectively targets mitochondrial metabolism in tumor cells by mobilizing lipoate-sensitive, cancer-specific regulation of PDH and KGDH, shutting down the two enzymes and thereby choking carbon flow through the TCA cycle. This attack on mitochondrial metabolism redundantly activates multiple cell death pathways (Zachar, et al., 2011, J Mol Med 89, 1137; Stuart, et al., 2014, Cancer&Metab 2, 4). In vitro, CPI-613 is uniformly effective in killing tumor cell lines (more than 50 tumor lines tested to date). Moreover, these drugs have produced compelling clinical responses (including complete remissions) in some, but not all patients with advanced, refractory, heavily pre-treated cancers in initial Phase I/II clinical trials (Pardee et al., 2014, Clin Cancer Res 20:5255). In an effort to explore the mechanisms of CPI-613 activity and characterize the reasons for differential patient responses, we have shown that the kinetics of the cancer cell death response to CPI-613 is profoundly affected by the carbon sources available to the cell (Zachar, et al., 2011, J Mol Med 89, 1137). For example, in media where pyruvate and glutamine (capable of producing net ATP synthesis only in the mitochondrial compartment) are the major carbon sources, death is rapid, completed within 4 hours (op cit). In contrast, when glucose is substituted for pyruvate, the cell death programs take up to 48 hours to complete. We have analyzed the mechanistic basis of this and related observations, including steady state and flux metabolomics, pharmacological inhibitors of non-TCA metabolic pathways, and manipulation of media composition to systematically characterize these dramatic effects of nutrient availability on cancer cell drug response. Our results demonstrate that CPI-613 induced inactivation of tumor cell KGDH is modulated by the potency of mitochondrial redox detoxification (Stuart, et al., 2014, Cancer&Metab 2, 4). Given that various metabolic pathways (for example, glycolysis) can ultimately contribute to mitochondrial ROS detoxification potential, it appears that substrate availability powerfully influences the cellular response to CPI-613. These insights open a number of fundamentally new approaches to increasing the fraction of patients who respond strongly to CPI-613 in the clinic. We will also report pre-clinical studies targeted at allowing planned future clinical trials to exploit these new insights. Citation Format: Shawn D. Stuart, Moises Guardado, Michael Dahan, Paul M. Bingham, Zuzana Zachar. Tumor metabolic remodeling can modulate anticancer drug response: CPI-613 attack on tumor cell mitochondrial metabolism is mediated by metabolite availability. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A61.

Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells.

Predictions about the cellular efficacy of drugs tested in vitro are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed in vitro. The Raman studies detect large panitumumab-induced differences in vitro in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, in vitro vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies.

Regulation of sensitivity of tumor cells to antitubulin drugs by Cdk1-TAZ signalling

Antitubulin drugs are commonly used for the treatment of numerous cancers. However, either the intrinsic or acquired resistances of patients to these drugs result in the failure of the treatment and high mortality of cancers. Therefore, identifying genes or signalling pathways involved in antitubulin drug resistances is critical for future successful treatment of cancers.TAZ (Transcriptional coactivator with PDZ-binding motif), which is a core component of the Hippo pathway, is overexpressed in various cancers. We have recently shown that high levels of TAZ in cancer cells result in Taxol resistance through up-regulation of downstream targets Cyr61 and CTGF. However, how TAZ is regulated in response to Taxol is largely unknown. In this study, we found that Cdk1 (Cyclin-dependent kinase 1) directly phosphorylated TAZ on six novel sites independent of the Hippo pathway, which further resulted in TAZ degradation through proteasome system. Phosphorylation-mimicking TAZ mutant was unstable, and therefore abolished TAZ-induced antitubulin drug resistances. This study provides first evidence that Cdk1 is a novel kinase phosphorylating and regulating TAZ stability and suggests that Cdk1-TAZ signalling is a critical regulator of antitubulin drug response in cancer cells and may be a potential target for the treatment of antitubulin-drug resistant cancer patients.

Abstract 5322: Collections of simultaneously altered genes as highly predictive markers of cancer cell drug response

Abstract Introduction: Many genetic markers of drug response have been reported in cancer pharmacogenomics studies. However, most of these markers fail to achieve clinical utility because they capture response in only a small fraction of the patient population; this shortcoming might result from a reliance on single-gene markers in contemporary personalized medicine. While there is growing evidence that therapeutic response can be modulated by the concerted impact of multiple genetic alterations, the combinatorics associated with many-body interactions has mostly prohibited the discovery of multi-gene biomarkers, even using computational methods. We developed a computational approach, MOCA (Multivariate Organization of Combinatorial Alterations), to partially address these challenges. Extending our method that accounts for pairwise interactions, MOCA combines many genomic alterations into biomarkers of drug response, using Boolean set operations coupled with optimization; in this framework the union, intersection, and difference Boolean set operations are proxies of molecular redundancy, synergy, and interference. The algorithm is fast, broadly applicable to genomics data, of immediate utility for prioritizing cancer pharmacogenomics experiments, and recovers known clinical findings without bias. Furthermore, results from this work connect many important, previously isolated observations. Major Findings: When applied to 416 pharmacogenomically characterized cancer cell lines from the Cancer Cell Line Encyclopedia, MOCA identified many known and potential markers of drug response. For example, correlation of ERBB inhibitor response drastically increased when considering EGFR (ERBB1), ERBB2, ERBB3, ERBB4, and KRAS alterations in a single marker. Similarly, a marker combining IGF1, IGF1R, and RAD51 alterations drastically increased correlation with IGF1R inhibitor response, relative to any of these three genetic markers considered in isolation. This approach is also powerful for determining subsets of gene-specific mutations that increase correlation with drug response. For instance, MOCA accurately captures the differential EGFR inhibitor response conferred by known activating EGFR mutations. Similarly, we find specific HDAC1 mutations cooperate with HDAC5 overexpression to potentiate cells to the HDAC inhibitor panobinostat. Additionally, considering all pairwise gene-drug interactions, MOCA recovers known and compelling correlations, including: RTK inhibitor resistance via c-MET, EGFR, ERBB2, and PDGFRB kinase switching; mutual exclusivity of TP53 mutation and response to the MDM2 inhibitor nutlin-3; greater nutlin-3 potentiation via MDM4, rather than MDM2, overexpression; MEK and RAF inhibitor response in BRAF mutated cell lines; and MEK inhibitor potentiating NRAS mutations. Masica, David L., & Rachel Karchin. Cancer Research 73 (2013): 1699-1708. Citation Format: David L. Masica, Rachel Karchin. Collections of simultaneously altered genes as highly predictive markers of cancer cell drug response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5322. doi:10.1158/1538-7445.AM2014-5322

Chapter Eleven - Single-Molecule Sensing of Caspase Activation in Live Cells via Plasmon Coupling Nanotechnology

Apoptotic caspases execute programmed cell death, where low levels of caspase activity are linked to cancer (Kasibhatla & Tseng, 2003). Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process; however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. To overcome this, we describe a procedure to develop peptide-linked gold nanoparticles that have unique optical properties and can serve as beacons to visualize the apoptotic drug response in cancer cells at the single-molecule level. By thorough analyses of their trajectories, one can reveal early-stage caspase-3 activation in live cells continuously and with no ambiguity.

Establishment of a human 3D lung cancer model based on a biological tissue matrix combined with a Boolean in silico model

For the development of new treatment strategies against cancer, understanding signaling networks and their changes upon drug response is a promising approach to identify new drug targets and biomarker profiles. Pre-requisites are tumor models with multiple read-out options that accurately reflect the clinical situation. Tissue engineering technologies offer the integration of components of the tumor microenvironment which are known to impair drug response of cancer cells. We established three-dimensional (3D) lung carcinoma models on a decellularized tissue matrix, providing a complex microenvironment for cell growth. For model generation, we used two cell lines with (HCC827) or without (A549) an activating mutation of the epidermal growth factor receptor (EGFR), exhibiting different sensitivities to the EGFR inhibitor gefitinib. EGFR activation in HCC827 was inhibited by gefitinib, resulting in a significant reduction of proliferation (Ki-67 proliferation index) and in the induction of apoptosis (TUNEL staining, M30-ELISA). No significant effect was observed in conventional cell culture. Results from the 3D model correlated with the results of an in silico model that integrates the EGFR signaling network according to clinical data. The application of TGFβ1 induced tumor cell invasion, accompanied by epithelial–mesenchymal transition (EMT) both in vitro and in silico. This was confirmed in the 3D model by acquisition of mesenchymal cell morphology and modified expression of fibronectin, E-cadherin, β-catenin and mucin-1. Quantitative read-outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model. The combined in vitro and in silico model represents a powerful tool for systems analysis.

Abstract 3848: Cryopreserved, viable and architecturally intact lung tissue from patients as a model to study drug therapies.

Abstract RATIONALE: Surgery, chemotherapy and radiation have not appreciably improved lung cancer patient survival rates over the past 20 years. Preclinical anti-tumor efficacy testing models could be improved by being rapid, reliable and capable of demonstrating tumor responses to dosing (concentration/timing) and drug interactions, while accounting for inter-patient variation. High-throughput, 3D in vitro systems hold promise as such therapeutic models, but linking these models to clinical responses in patients has been a dilemma. METHODS: We developed a lung tissue cryopreservation/banking method that optimizes cell viability and maintains tissue architecture after thawing. This method involves the expansion-perfusion of fresh patient lung tissue samples with non-toxic cryoprotectant solutions, followed by controlled-rate freezing of samples. RESULTS: We used this method to cryopreserve lung tissues from multiple patients. Although inter-patient variability in lung tissue compliance was anticipated, a wide range of expansion volumes can be tolerated without architectural damage. In contrast, tissue integrity was more sensitive to variations in expansion-perfusion rates (as gauged by lymphatic expansion/tearing). Cryosubstitution demonstrated that crystal formation varies with cryoprotectant cocktail and region within the lung, but can be virtually eliminated under certain conditions. From thawed lung tissues, we have 1) isolated and expanded viable lung cell types; 2) isolated decellularized extracellular components; and, 3) reconstituted intact lung tissue with fluorescent-tagged lung tumor cells. We performed patient surgeries/preservations individually, thawing, processing and analysis of lung tissue from multiple patients was performed simultaneously. CONCLUSION: Cancer cell drug responses are being compared in lung tissues ex vivo versus on standard monolayer culture using advanced proteo-/lipidomic mapping techniques (including MALDI MS and DIGE). Comparing multiple patient-specific lung tissues simultaneously should afford patient population stratification based on molecular profiles and achieve more rapid methods for determining personalized clinical treatment regiments. Citation Format: Demetri D. Spyropoulos, Chadrick E. Denlinger, Ellen C. Riemer, Danforth A. Newton, E. Ellen Jones, Richard R. Drake, John E. Baatz. Cryopreserved, viable and architecturally intact lung tissue from patients as a model to study drug therapies. [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 3848. doi:10.1158/1538-7445.AM2013-3848

Collections of Simultaneously Altered Genes as Biomarkers of Cancer Cell Drug Response

Computational analysis of cancer pharmacogenomics data has resulted in biomarkers predictive of drug response, but the majority of response is not captured by current methods. Methods typically select single biomarkers or groups of related biomarkers but do not account for response that is strictly dependent on many simultaneous genetic alterations. This shortcoming reflects the combinatorics and multiple-testing problem associated with many-body biologic interactions. We developed a novel approach, Multivariate Organization of Combinatorial Alterations (MOCA), to partially address these challenges. Extending on previous work that accounts for pairwise interactions, the approach rapidly combines many genomic alterations into biomarkers of drug response, using Boolean set operations coupled with optimization; in this framework, the union, intersection, and difference Boolean set operations are proxies of molecular redundancy, synergy, and resistance, respectively. The algorithm is fast, broadly applicable to cancer genomics data, is of immediate use for prioritizing cancer pharmacogenomics experiments, and recovers known clinical findings without bias. Furthermore, the results presented here connect many important, previously isolated observations.

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

Genetic Instability Influences Drug Response in Cancer Cells

One of the main reasons why most patients with advanced cancer are not curable with the therapies available is the broad heterogeneity of cancer cells, inherently related to their genomic instability that reflects defects of cell cycle checkpoints and DNA mismatch repair (MMR). The present paper reviews Today's knowledge of MMR. Microsatellite (DNA repetitive sequences) instability (MSI) used as a surrogate marker of MMR defects was associated with a predisposition to somatic mutations of several genes including those involved in the neoplastic transformation and tumor progression. Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by germ line mutation in genes involved in MMR such as hMLH1 or hMLH2, or less frequently hMLH6 or hPMS2; it is associated with a high risk of intestinal cancer (CRC) and other tumors including endometrial, stomach, kidney and brain. There is ample preclinical evidence that cells deficient in MMR are resistant to methylating agents and to some antimetabolites, including 5FU, which is the drug used most for the CRC, whereas they are equally sensitive to oxaliplatin and possibly more sensitive to irinotecan. More studies are needed on the importance of MMR for sensitivity to different anticancer regimens and drugs, so this knowledge can guide rational therapy according to the tumor MMR status.