Vulnerability Of Tumor Cells Research Articles

Abstract A90: Mesothelin confers tumor cell vulnerabilities for gefitinib treatment

Abstract Combination therapy for pancreatic adenocarcinoma (PDAC) has recently had two successful stage III trials with FOLFIRINOX (combination folinic acid, fluorouracil, irinotecan, and oxalplatin) and combination gemcitabine and paclitaxel. Because of the heterogeneity of PDAC, the benefits of combination therapy are immediately apparent since it targets multiple facets of cancer simultaneously. However, FOLFIRINOX and gemcitabine + paclitaxel are highly toxic and affect all dividing cells, making apparent the need for better understanding of the cell profiles in PDAC. The epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many cancers, with EGFR or its ligands often overexpressed or mutated. Recently we have found that EGFR expression is correlated to mesothelin (MSLN) expression in PDAC. MSLN, a cell surface glycoprotein, has been shown by us and other labs to be overexpressed in 80 to 90% of all PDAC cases. To examine the relationship between MSLN and EGFR, we modulated MSLN expression by generating a MSLN overexpressing cell line from MIA-PaCa2 cells, and a MSLN knockdown cell line from CAPAN-2 cells. The change in EGFR signaling mimicked that of MSLN, with MSLN overexpression significantly increasing EGFR expression and MSLN knockdown significantly decreasing EGFR expression. Furthermore, high MSLN (and therefore high EGFR expression) showed increased sensitivity to gefitinib, an EGFR ATP binding site inhibitor, at concentrations as low as 100 nM, while MSLN low expressing cells did not exhibit any effect from gefitinib at concentrations below 10 µM. Cell Cycle analysis demonstrated that gefitinib increased the number of cells in G1 phase after 24 hours in MSLN high expressing cells, but had no effect on MSLN low expressing cells. In addition, western blot analysis of EGFR downstream pathways showed an increase in phosphorylated phospholipase C-Υ (PLC-Υ) in MSLN high expressing cells, which could be mitigated by treatment with gefitinib. Finally, combination therapy with gemcitabine and 100 nM gefitinib showed an additive effect in MSLN high expressing cells, while in MSLN low expressing cells combination gemcitabine and gefitinib had no additional effect over gemcitabine alone. This study demonstrates the correlation between MSLN expression and EGFR expression and indicates that gefitinib could be a potential addition to combination therapy for PDAC in patients with high MSLN expression. Citation Format: Ethan Poteet, Zhengdong Liang, Youwen Wang, Changyi Chen, Qizhi Yao.{Authors}. Mesothelin confers tumor cell vulnerabilities for gefitinib treatment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A90.

Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation ofcytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

Abstract B43: PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment

Abstract B43: PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment

Cupriphilic compounds to aid in proteasome inhibition

It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6,7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3μM in the presence of 100nM copper.

Tumor-selective mitochondrial network collapse induced by atmospheric gas plasma-activated medium.

Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. However, the mechanisms of its tumor-selective cytotoxicity remain unclear. Here we report that AGP-activated medium increases caspase-independent cell death and mitochondrial network collapse in a panel of human cancer cells, but not in non-transformed cells. AGP irradiation stimulated reactive oxygen species (ROS) generation in AGP-activated medium, and in turn the resulting stable ROS, most likely hydrogen peroxide (H2O2), activated intracellular ROS generation and mitochondrial ROS (mROS) accumulation. Culture in AGP-activated medium resulted in cell death and excessive mitochondrial fragmentation and clustering, and these responses were inhibited by ROS scavengers. AGP-activated medium also increased dynamin-related protein 1-dependent mitochondrial fission in a tumor-specific manner, and H2O2 administration showed similar effects. Moreover, the vulnerability of tumor cells to mitochondrial network collapse appeared to result from their higher sensitivity to mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment.

CBIO-31MECHANISMS OF RESISTANCE TO EGFR INHIBITION REVEAL METABOLIC VULNERABILITIES IN HUMAN GBM

Therapeutic targeting of EGFR signaling in GBM has had largely disappointing results in the clinic. We sought to identify cell vulnerabilities that could be targeted in combination with EGFR. Using a murine model for GBM, primary patient-derived xenografts (PDXs), and human tumor tissue, we identify cellular metabolic changes as an important mechanism of EGFR resistance. Rather than acquired resistance, we observe a subpopulation of tumor cells with unique properties, including increased 3D tumor cell invasion, expression of progenitor cell markers, and altered lipid metabolism, that are uniquely poised to resist EGFR inhibition. Interestingly, this subpopulation of cells is selectively vulnerable to changes in lipid metabolism suggesting its importance in survival. Selection for this subpopulation appears specific to EGFR inhibition, as it is not conferred by inhibition of pathways downstream of EGFR, including Src, PI3K/Akt, and MAPK. In certain human tumors and GBM PDX, expression levels of lipid metabolic enzymes is high in the absence of EGFR inhibition. In these tumors enzyme inhibition conferred decreased tumor cell survival and invasion. Preclinical studies will evaluate the benefit of inhibiting lipid metabolic enzymes in PDX. These data identify specific tumor cell vulnerabilities and suggest possible strategies to target EGFR resistance mechanisms.

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

Abstract 3837: Super-enhancers define breast cancer subclasses and identify novel tumor cell vulnerabilities

Abstract Breast cancer cell state is driven by genomic mutational events, aberrant signaling cascades and transcriptional misregulation. The profound clinical impact of ER-modulatory drugs underscores the importance of understanding and targeting the transcriptional dependencies of breast cancer cells in an effort to create new therapeutics. Recently, large clusters of cis-acting enhancers, termed Super-enhancers, have emerged as dominant transcriptional regulators of oncogenes and other key tumor drivers in cancer cells, indicating that these elements control and maintain breast tumor cell state. Mapping these elements and the genes they control in patient tumors can provide insight into breast cancer classification and identify therapeutic targets. We have discovered Super-enhancers in breast cancer patient tissue and have characterized their roles in enforcing tumor cell state. We find that Super-enhancers define known clinical subgroups in invasive ductal carcinoma and pinpoint the putative transcriptional drivers of each subgroup. Super-enhancer-associated genes encode both known and novel therapeutic targets including kinases, phosphatases, chromatin regulators and transmembrane proteins, including key drivers such as ERRB2 in HER2+ patient samples, ESR1 in estrogen receptor positive samples, and CCND1 in samples of luminal subtype. shRNA knockdown confirms that Super-enhancer regulated genes are indeed essential for tumor cell survival and that Super-enhancer analysis in primary patient samples can be used to define new subtype specific cancer vulnerabilities. In sum, we describe the biological and disease relevance of Super-enhancer-associated genes in the context of tumor cell state, genome mutational landscape and drug target discovery. Citation Format: Cindy Collins, Mei Wei Chen, Matthew Eaton, David Orlando, Michael McKeown, Christian Fritz, Eric Olson, Matthew Guenther. Super-enhancers define breast cancer subclasses and identify novel tumor cell vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2015-3837

Abstract LB-249: TDG, a dual genomic and epigenomic regulator, as a novel antimelanoma target

Abstract Melanoma is an aggressive cancer resistant to treatment, whose incidence has increased over the past two decades. Although the majority of melanoma cases are cured after surgical excision of the primary tumor, metastases occur frequently, and the metastatic form of the disease has a poor prognosis and is highly resistant to all current forms of therapy. Thus, new prognostic factors and advanced therapeutic strategies are urgently needed. We recently reported that the base excision repair protein Thymine DNA Glycosylase (TDG) has dual roles in safeguarding the genome and the epigenome (Cell 146:67, 2011). TDG not only protects CpG sites from spontaneous deamination of 5-methylcytosine and cytosine (genomic stability), but importantly, at the epigenomic level, acts in a DNA demethylation pathway that converts 5-methylcytosine to cytosine (epigenomic stability). Specifically TDG removes the novel bases 5-formylcytosine and 5-carboxylcytosine, demethylation intermediates produced by the upstream TET dioxygenases. TET alterations have been recently found in melanoma and correlate with poor prognosis. Moreover, TDG sequence variants in melanoma are reported in the TCGA database. For these reasons, we began studying the functional significance of TDG in melanoma. We reasoned that the two non-redundant (genomic and epigenomic) functions of TDG may represent a vulnerability of tumor cells and be exploited as novel drug targets for cancer treatment, because targeting TDG would achieve the dual effect of impairing DNA repair and disrupting the epigenetic state of the cancer cell. We found that reduced TDG levels correlate with tumorigenic melanomas and therefore TDG inhibition might further promote aggressiveness. Unexpectedly, however, TDG knockdown in melanoma lines caused cell cycle arrest, senescence and ultimately cell death. Senescence and cell death induced by TDG knockdown occurred without apparent activation of the DNA damage response, based on absence of H2AX phosphorylation. These in vitro findings were confirmed in vivo, as TDG knockdown in melanoma lines blocked tumor formation in xenografts. Given its potential as a novel therapeutic target, we conducted a pilot high-throughput screen and identified first-generation TDG chemical inhibitors. Two compounds were confirmed to inhibit TDG repair activity in vitro by radioactive-based glycosylase assay. Importantly, both inhibitors also blocked TDG demethylase function in cells, as evidenced by increased staining intensity of 5-carboxylcytosine. Both compounds inhibited proliferation (by clonogenic, MTT and Xcelligence assays) of melanoma cell lines in the micromolar range and could synergize with alkylating agents and other anti-melanoma drugs. Thus, while reduced TDG levels may be part of the tumorigenesis process, limited levels of TDG are essential for melanoma viability. Therefore, TDG inhibition may represent a novel approach for melanoma treatment. Citation Format: Rossella Tricarico, Pietro Mancuso, Vikram Bhattacharjee, Neil Beeharry, Emmanuelle Nicolas, Margret Einarson, Laura Cosentino, Irwin Davidson, Lionel Larue, Robert W. Sobol, Timothy J. Yen, Alfonso Bellacosa. TDG, a dual genomic and epigenomic regulator, as a novel antimelanoma target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-249. doi:10.1158/1538-7445.AM2015-LB-249

Therapeutic targeting of cellular metabolism in cells with hyperactive mTORC1: a paradigm shift.

mTORC1 is an established master regulator of cellular metabolic homeostasis, via multiple mechanisms that include altered glucose and glutamine metabolism, and decreased autophagy. mTORC1 is hyperactive in the human disease tuberous sclerosis complex (TSC), an autosomal dominant disorder caused by germline mutations in the TSC1 or TSC2 gene. In TSC-deficient cells, metabolic wiring is extensively disrupted and rerouted as a consequence of mTORC1 hyperactivation, leading to multiple vulnerabilities, including "addiction" to glutamine, glucose, and autophagy. There is synergy between two rapidly evolving trajectories: elucidating the metabolic vulnerabilities of TSC-associated tumor cells, and the development of therapeutic agents that selectively target cancer-associated metabolic defects. The current review focuses on recent work supporting the targeting of cellular metabolic dysregulation for the treatment of tumors in TSC, with relevance to the many other human neoplasms with mTORC1 hyperactivation. These data expose a fundamental paradox in the therapeutic targeting of tumor cells with hyperactive mTORC1: inhibition of mTORC1 may not represent the optimal therapeutic strategy. Inhibiting mTORC1 "fixes" the metabolic vulnerabilities, results in a cytostatic response, and closes the door to metabolic targeting. In contrast, leaving mTORC1 active allows the metabolic vulnerabilities to be targeted with the potential for a cytocidal cellular response. The insights provided here suggest that therapeutic strategies for TSC and other tumors with activation of mTORC1 are at the verge of a major paradigm shift, in which optimal clinical responses will be accomplished by targeting mTORC1-associated metabolic vulnerabilities without inhibiting mTORC1 itself.

MAP3K1: Genomic Alterations in Cancer and Function in Promoting Cell Survival or Apoptosis

MAP3K1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. MAP3K1 regulates JNK activation and is unique among human kinases in that it also encodes an E3 ligase domain that ubiquitylates c-Jun and ERK1/2. Full length MAP3K1 regulates cell migration and contributes to pro-survival signaling while its caspase 3-mediated cleavage generates a C-terminal kinase domain that promotes apoptosis. The critical function of MAP3K1 in cell fate decisions suggests that it may be a target for deregulation in cancer. Recent large-scale genomic studies have revealed that MAP3K1 copy number loss and somatic missense or nonsense mutations are observed in a significant number of different cancers, being most prominent in luminal breast cancer. The alteration of MAP3K1 in diverse cancer types demonstrates the importance of defining phenotypes for possible therapeutic targeting of tumor cell vulnerabilities created when MAP3K1 function is lost or gained.

Abstract A27: Dissecting the functional landscape of triple-negative breast cancer

Abstract Our group employed a pangenomic loss of function screening approach to systematically dissect the molecular architecture that functionally supports Triple Negative Breast Cancer (TNBC). Given that treatment options for this disease are typically limited to cytotoxic therapies, we applied a paclitaxel-based synthetic lethal approach to identify those molecular components that modulate chemoresponsiveness. To account for heterogeneity within the patient population, we screened 4 triple negative tumor-derived breast cancer cell lines that represent the spectrum of TNBC oncogenic aberrations and chemoresponse profiles. We also accounted for the two molecular subtypes, Claudin-Low and Basal-Like, that comprise the majority of TNBC cases. These screens revealed a number of core modulators of tumor cell viability and paclitaxel-induced cellular stress that have not been previously appreciated for supporting TNBC biology at the cell autonomous level. For example, this strategy implicated AMPK family member, SIK2, as a key nutrient sensor that may inhibit excessive autophagy. Inhibition of SIK2 leads to enhanced autophagic flux and a loss of cell viability in a variety of TNBC genetic backgrounds. In addition, we discovered the signaling supported by the cytokine receptor, CXCR3, and its ligand, CXCL9, to be repurposed to promote mitotic fidelity and thus, tumor cell fitness specifically in the Basal-Like TNBC molecular subtype. Finally, we uncovered a requirement for the transcription factor, ZNF165, in specifying the gene expression profiles activated by TGFβ signaling. In particular, the TGFβ target protein, SERPINE1, is required to promote prosurvival signaling in the Claudin-Low TNBC molecular subtype. Given that the expression of ZNF165 is otherwise restricted to the male testes, ZNF165 may represent a mechanism by which tumors engage anomaly-expressed proteins to promote survival. Taken together, we have uncovered novel tumor cell vulnerabilities in TNBC that could be therapeutically leveraged to enhance current therapeutic approaches. Citation Format: Kimberly Maxfield, Aleix Prat, Kathleen Corcoran, Angelique Whitehurst. Dissecting the functional landscape of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A27.

Abstract LB-207: Dissecting the functional landscape of triple-negative breast cancer.

Abstract Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor patient prognosis. Patients who respond poorly to the current standard of care cytotoxic chemotherapeutic regimens only have about a 55% five-year survival rate due to the lack of other effective TNBC therapeutics. To uncover new therapeutic entry points for TNBC, we employed a pangenomic loss of function siRNA screening approach. To account for heterogeneity within the patient population, we screened 4 triple negative tumor-derived breast cancer cell lines that represent the spectrum of oncogenic aberrations and molecular subtypes in TNBC. By intersecting the datasets from these 4 cell lines, we revealed core modulators of viability and chemotherapy-induced cellular stress that have not been previously implicated in TNBC biology. For example, our screen uncovered a tractable energy-sensing AMPK family member that supports tumor cell viability in a subset of TNBC tumors. We also discovered tumor cell vulnerabilities unique to the Basal-Like and Claudin-Low TNBC molecular subtypes. In particular, Basal-Like tumor cell division is sensitive to perturbations in specific cytokine signaling. With respect to Claudin-Lows, we found an increased sensitivity to depletion of transcriptional modulators and downstream targets of TGFβ signaling, which suggests that TGFβ signaling may not only be required for a mesenchymal phenotype but also for Claudin-Low tumor cell survival. Our findings suggest that a set of tumor cell vulnerabilities can partition TNBC into functional subgroups and these vulnerabilities could lead to new therapeutics improving overall TNBC patient prognosis. Citation Format: Kimberly Maxfield, Aleix Prat, Kathleen Corcoran, Angelique Whitehurst. Dissecting the functional landscape of triple-negative breast cancer. [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 LB-207. doi:10.1158/1538-7445.AM2013-LB-207

Abstract 5185: RNAi screen identifies a synthetic lethal interaction between Pim1 overexpression and Plk1 inhibition.

Abstract PIM1 kinase is overexpressed in many tumor types including lymphomas and prostate cancer, where it is known to cooperate with the MYC oncogene in promoting tumorigenicity. PIM1 enhances MYC stability and transcriptional activity. Knowledge of the vulnerabilities of Pim1-expressing tumor cells will be of great value in efforts to develop novel anti-cancer therapeutics. Here we have used RNAi screening to identify genes whose depletion is detrimental to Pim1-overexpressing cells. We screened a collection of 570 siRNAs targeting cell cycle, apoptosis, serine-threonine kinase and tyrosine kinase genes for their effects on the viability of Pim1-expressing RWPE1 prostate epithelial cells. We identified the mitotic regulator polo-like kinase (PLK1) as a gene whose depletion is particularly detrimental to the viability of Pim1-expressing prostate cancer cells in vitro and in vivo. Inhibition of PLK1 by shRNA or by the chemical inhibitor BI2536 in prostate cancer xenograft model resulted in a dramatic inhibition of tumor progression in Pim1-overexpressing cells. Interestingly Pim1-overexpressing cells are more prone to mitotic arrest followed by apoptosis. Furthermore, inhibition of Plk1 activity led to the reduction of MYC protein levels both in vitro and in vivo. These results suggest that PLK1 inhibition may be particularly effective against PIM1-expressing tumors due, at least in part, to the fact that PLK1 inhibition reduces MYC protein levels. Citation Format: Sarki Abdulkadir, Riet Vander Meer, Meejeon Roh. RNAi screen identifies a synthetic lethal interaction between Pim1 overexpression and Plk1 inhibition. [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 5185. doi:10.1158/1538-7445.AM2013-5185 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Multiple Cancer Testis Antigens Function To Support Tumor Cell Mitotic Fidelity

While the expression of genes that are normally involved in spermatogenesis is frequently detected in tumors, the extent to which these gene products are required for neoplastic behaviors is unclear. To begin to address their functional relevance to tumorigenesis, we identified a cohort of proteins which display synthetic lethality with paclitaxel in non-small-cell lung cancer and whose expression is biased toward testes and tumors. Remarkably, these testis proteins, FMR1NB, NXF2, MAGEA5, FSIP1, and STARD6, are required for accurate chromosome segregation in tumor cells. Their individual depletion enhances the generation of multipolar spindles, increases mitotic transit time, and induces micronucleation in response to an otherwise innocuous dose of paclitaxel. The underlying basis for abnormal mitosis is an alteration in microtubule function, as their depletion increases microtubule cytaster formation and disrupts microtubule stability. Given these observations, we hypothesize that reactivated testis proteins may represent unique tumor cell vulnerabilities which, if targeted, could enhance responsiveness to antimitotic therapy. Indeed, we demonstrate that combining paclitaxel with a small-molecule inhibitor of the gametogenic and tumor cell mitotic protein TACC3 leads to enhanced centrosomal abnormalities, activation of death programs, and loss of anchorage-independent growth.

Eukaryotic Initiation Factor 4F: A Vulnerability of Tumor Cells

Protein synthesis is a complex, tightly regulated process in eukaryotic cells and its deregulation is a hallmark of many cancers. Translational control occurs primarily at the rate-limiting initiation step, where ribosomal subunits are recruited to template mRNAs through the concerted action of several eukaryotic initiation factors (eIFs). One factor that interacts with both the mRNA and ribosomes, and appears limiting for translation is eIF4F, a complex composed of the cap-binding protein, eIF4E; the scaffold protein, eIF4G; and the ATP-dependent DEAD-box helicase, eIF4A. eIF4E appears to play an important role in tumor initiation and progression since its overexpression can cooperate with oncogenes to accelerate transformation in cell lines and animal models, and its levels are elevated in many human cancers. This, therefore, represents a vulnerability for transformed cells, and presents an opportunity for therapeutic intervention. In this review, we discuss approaches for targeting eIF4F activity.

Combination Therapy of an Inhibitor of Group VIA Phospholipase A 2 with Paclitaxel Is Highly Effective in Blocking Ovarian Cancer Development

We and others have shown that calcium-independent phospholipase A(2) (iPLA(2)) is involved in epithelial ovarian cancer (EOC). Hence, we propose that iPLA(2) is a potential effective and novel target for EOC. We tested this concept and found that bromoenol lactone (BEL), a selective inhibitor of iPLA(2), significantly inhibited EOC metastatic tumor growth in mouse xenograft models using human SKOV3 and HEY ovarian cancer cells. Moreover, the combination of BEL with paclitaxel (PTX), one of the most commonly used therapeutic agents in EOC, almost completely blocked tumor development in the xenograft mouse model. BEL showed no detectable cytotoxic effects in mice. Another iPLA(2) inhibitor, FKGK11, also inhibited tumor development in the xenograft mouse model, supporting that the major target of action was iPLA(2). The additional effects of BEL with PTX in vivo likely stem from their distinct cellular effects. BEL and FKGK11 reduced adhesion, migration, and invasion of EOC cells in vitro; the reduced ability to adhere, migrate, and invade seems to increase the vulnerability of tumor cells to PTX. These results provide an important basis for the development of new treatment modalities for EOC.

Abstract 994: Monitoring glucose, glutamine and fatty acid metabolism to determine metabolic vulnerability of tumor cells in vitro

Abstract Tumor cells display a metabolism that is strikingly different from that of the normal tissue from which they originated. This metabolic alteration results from activation of oncogenes and/or loss of tumor suppressor genes controlling cellular metabolic pathways as well as adaptation to their microenvironments and provides energy and cellular building blocks, allowing tumor cells to proliferate and survive. The altered metabolism of glucose and glutamine are becoming one of the hallmarks of tumor cells. We present a method that enables rapid assessment of the ability of tumor cells to catabolize the three major nutrient substrates, glucose, glutamine and fatty acids in a microplate using the XF Extracellular Flux Analyzer. Cellular oxygen consumption rate is used as an indicator of substrate oxidation by the mitochondrial respiratory chain upon substrate addition and extracellular acidification rate upon glucose addition is used as an indicator of glycolysis. Furthermore, the maximal capacity of glycolysis and of mitochondrial substrate oxidation is determined by forcing one of the two energy-producing pathways to operate at maximum. In addition, pharmacological inhibition of distinct metabolic pathways allows us to discern the biochemical pathways used, i.e., whether glutamine is metabolized through aminotransferase or glutamate dehydrogenase. Using this approach, we have demonstrated that the nutrient levels in culture medium can markedly shift the ability of cancer cells to use glucose for glycolysis versus oxidation of glutamine and fatty acids and is accompanied by modulation of cell proliferation rate. For example, excessive nutrients induced activation of fatty acid oxidation and enhanced glutamine oxidation in myc-expressing SF188 glioblastoma cells. This shift in substrate use also sensitized these cells to inhibition of fatty acid oxidation. We observed a concomitant increase in the maximal capacity of the mitochondrial respiratory chain with elevated substrate oxidation. In contrast, the apparent glycolysis rate of SF188 cells was much reduced but the glycolytic capacity was retained, which implies these cells have the flexibility to channel glucose to either energy or building block production required for cell mass duplication. We suggest that this type of dynamic analysis provides a rapid means to determine the ability of various types of tumor cells to use glucose, glutamine and fatty acids as well as the maximal capacity of the machineries of glycolysis and substrate oxidation, which may be indicative of metabolic vulnerability and therefore targets of intervention of a particular tumor cell type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2011-994

Abstract 3525: Targeting Group VIA phospholipase A2 using small molecules for ovarian cancer treatment

Abstract Epithelial ovarian cancer (EOC) causes estimated 13,850 deaths in 2010 in the United State. More effective treatment regimens are urgently required for this deadly disease. We and others have obtained ample evidence indicating that lysophosphatidic acid (LPA) plays an important role in EOC development. We have recently shown that the levels of LPA and several other bioactive lipids in the tumor environment of EOC are regulated by Group VIA calcium-independent phospholipase A2 beta (iPLA2β) expressed in tumor microenvironment, suggesting that targeting iPLA2β in both host and tumor cells will likely to be beneficial. To test this idea, we used bromoenol lactone (BEL), a selective inhibitor of iPLA2β in vivo, and found that BEL significantly inhibited EOC tumorigenesis and metastasis in mouse xenograft models using human SKOV3 and HEY ovarian cancer cells. Moreover, the combination of BEL with paclitaxel (PTX), one of the mostly commonly used therapeutics in EOC, almost completely blocked tumor development in the xenograft mouse model. BEL showed very low or no detectable cytotoxic effects in mice. Another iPLA2 inhibitor, FKGK11, also inhibited tumor development in the xenograft mouse model, supporting that the major target of action was iPLA2. The combinational effects of BEL and PTX in vivo likely stem from their distinct cellular effects. BEL and FKGK11 reduced adhesion, migration, and invasion of EOC cells in vitro; the reduced ability appears to increase the vulnerability of tumor cells to PTX. In summary, this is the first attempt to target iPLA2 using a small inhibitor molecule for cancer treatment in vivo. In addition, the work is also rather unique by targeting activity of the same molecule (iPLA2) in both host and tumor cells. These results provide an important basis for development of new treatment modalities for EOC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3525. doi:10.1158/1538-7445.AM2011-3525

Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis.

Pretreatment of the human melanoma cell line, A375, and the human colon carcinoma cell line, HT-29, with certain cytokines was found to increase the vulnerability of these cells to monocyte-mediated killing. This activity was found to correlate with increased expression of intercellular adhesion molecule-1 (ICAM-1) on the tumor cells and was blocked by anti-ICAM-1 antibodies. Both IFN-gamma and TNF induced large increases in the ICAM-1 expression on both cell lines and increased the susceptibility of the tumor cells to monocyte-mediated killing. IFN-alpha and IL-1 beta, however, induced only small increases in ICAM-1 expression and enhanced the lysis of the A375 cells but not the HT-29 cells by monocytes. These differences may be the result of a higher basal expression of ICAM-1 found on the A375 cells when compared with the HT-29 cells. These data indicate that regulation of ICAM-1 expression on tumor cells can alter the vulnerability of these cells to lysis by monocytes.