Browsing by Subject "Carcinoma, Non-Small-Cell Lung"
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Item 6-year Pulmonary Lobectomy Analysis Comparing Robotic to Thoracotomy and VATS: Impact to a State University Cardiothoracic Training Program(2017-01-17) Nawalaniec, James; Kernstine, Kemp; Elson, Matthew; Yuan, Chaofan; Madrigales, Alejandra; Lysikowski, JerzyOBJECTIVE: Current understanding of robotic lobectomy is largely from established thoracic surgical programs, often single-surgeon experience and does not involve trainees. Our objective is to assess the effect that a new robotic lobectomy program might have on a state university cardiothoracic (CT) training program to patient care, CT training, and the institution. METHODS: Our IRB-approved, prospectively maintained database and query of anatomic lung resections between 1/1/2006 to 6/30/2016 was verified with the medical center EMR and further verified with blinded double-entry. Inclusion criteria required a documented anatomic resection. Cost and oncologic data was obtained from the analytics department and tumor registry, respectively. Propensity scores were assigned based on age, sex, and five comorbidities. Lung cancer survival was analyzed by the Kaplan-Meier method and compared to the SEER database. Our robotic CT training method consists of a 6-month program over 3 years; the first 3 months focus on simulation and bedside-assist and the last 3 months, on complete case set-up and console training; adjustments made dependent upon the trainees' prior minimally invasive experience. RESULTS: 523 consecutive cases were identified, 91 cases were excluded. The query identified 212 robotic (179 non-small cell lung cancer (NSCLC)), 160 thoracotomy (117 NSCLC) and 60 video-assisted (VATS) (44 NSCLC) cases. Multiple surgeons performed each approach. Operative results and clinical outcomes favored robotic surgery compared to thoracotomy and showed little difference with VATS. Robotics increased lymph node stations, nodes, and upstaging rates, with similar R0 compared to thoracotomy and VATS; there was no significant difference in survival. A CT resident served as the console surgeon in 35% of all cases: 0% in the first two years increasing to 79% in the latest year. Minimally invasive surgeries increased from 32% of all cases in the first year of robotics to 89% in the latest year. The total volume of lung cancer treated at the center increased by 51%, surgical cases by 220%, and clinical trial accrual by 92%, since introducing robotics. CONCLUSION: A robotic surgery training component can be implemented at a state university cardiothoracic training program without sacrificing quality. Robotic surgery in this setting offers the same or better clinical results, is cost-effective, and is oncologically-sound. Additionally, a robotic program may increase an institution's lung cancer volume, enhancing both the CT resident training experience and clinical research. This analysis has also identified opportunities to further improve efficiency and reduce cost.Item Analysis of ASCL1 in Neuroendocrine Lung Cancer(2015-08-20) He, Min; Buszczak, Michael; Cobb, Melanie H.; White, Michael A.; Liu, QinghuaSmall cell lung cancer (SCLC) is an understudied tumor subset with aggressive neuroendocrine carcinoma features. Previous studies have determined that the basic helix-loop-helix (bHLH) transcription factor achaete-scute homolog 1 (ASCL1) is essential for the survival and progression of many pulmonary neuroendocrine (NE) cancer cells, which include both SCLC and some non-small cell lung cancer (NSCLC). To understand how ASCL1 initiates tumorigenesis in pulmonary neuroendocrine cancer and identify the transcriptional targets of ASCL1, whole-genome RNA-sequencing (RNA-seq) analysis combined with chromatin immunoprecipitation-sequencing (ChIP-seq) were performed with a series of lung cancer cell lines. We discovered the gene SCNN1A, which encodes the alpha subunit of the epithelial sodium channel (αENaC), is highly correlated with ASCL1 expression in SCLC cells. We confirmed that SCNN1A is under the transcriptional control of ASCL1, indicating that SCNN1A represents a newly recognized ASCL1 target. The product of the SCNN1A gene ENaC can be pharmacologically inhibited by amiloride, a drug that has been used clinically for nearly 50 years. Amiloride-treated ASCL1-dependent tumor cells stopped cell growth in vitro. Analysis of downstream targets of ASCL1 broadens our understanding how ASCL1 functions, and further provides a step forward in the development of drug-targeted therapy for pulmonary neuroendocrine cancer. We also discovered that ASCL1 may negatively regulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through a negative feedback mechanism. Finally, we found that expression of ASCL1 in certain NSCLC could induce neuroendocrine features which are reminiscent of SCLC.Item Anti-VEGF Induced Reduction in Microvessel Density Does Not Correlate with Anti-Tumor Repsonse in Lung Cancer Xenografts(2013-01-22) Jacob, Antonia J.; Sullivan, Laura A.; Toombs, Jason E.; Minna, John D.; Brekken, Rolf A.Vascular endothelial growth factor-A (VEGF) is a primary stimulant of angiogenesis in pathological conditions including tumor progression. Strategies to block VEGF activity prevent or slow tumor growth in preclinical settings; however, clinical studies with bevacizumab, a monoclonal antibody (mAb) specific for VEGF have resulted in only modest benefit to a subset of patients with lung cancer. Previous studies in our laboratory defined the therapeutic efficacy of bevacizumab and an alternative anti-VEGF mAb (r84) in 12 non-small cell lung cancer (NSCLC) xenografts. Three NSCLC xenografts (Calu-6, A549 and Calu-3) showed intrinsic resistance to bevacizumab therapy. In the present study we evaluated whether microvessel density (MVD) could be used to 1) demonstrate if the anti-VEGF mAbs were effective at reducing VEGF-driven angiogenesis and 2) if MVD changes induced by bevacizumab or r84 correlated with overall therapeutic efficacy as determined by tumor size after chronic therapy. 3-5 tumors from animals bearing NSCLC xenografts treated with a control mAb (XTLl, bevacizumab or r84 were evaluated by immunohistochemistry for endothelial cells as a measure of microvessel density. Two independent endothelial cell markers were used, endomucin and CD31. In 11 of the 12 xenografts treatment with bevaclzumab or r84 significantly reduced MVD compared to XTL treatment, suggesting that bevacizumab and r84 do reduce VEGF-driven angiogenesis. However, the reduction in MVD induced by anti-VEGF therapy did not correlate with overall tumor response to therapy. These results strongly implicate resistance to anti-VEGF therapy is not mediated by activation af alternative angiogenic programs to compensate for VEGF blockade. Further the results suggest that tumor cell adaptation to therapy-induced hypoxia underlies poor therapeutic response to anti-VEGF strategies. Microarray of gene expression analysis of control treated tumors revealed several genes associated with metabolism, proliferation, and metastasis were significantly increased in tumors that displayed intrinsic resistant to bevacizumab. We conclude that response of tumor cells to therapy-induced hypoxia is a critical feature that drives the overall efficacy of anti-VEGF strategies.Item AXL Targeting Restores PD-1 Blockade Sensitivity of STK11/LKB1 Mutant NSCLC Through Expansion of TCF1+ CD8 T Cells(December 2021) Li, Huiyu; Akbay, Esra A.; DeBerardinis, Ralph J.; Fu, Yang-Xin; Brekken, Rolf A.; Minna, John D.; Aguilera, Todd A.Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB) for unknown reasons. We found that introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically, this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. We found that systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expands tumor-associated TCF1+ PD-1+ CD8 T cells, restoring therapeutic response to PD-1 ICB for KPL tumors. This effect was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. Anecdotal NSCLC patients with STK11/LKB1 mutant tumors also demonstrated responses to the combination of AXL inhibitor bemcentinib and pembrolizumab. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.Item Beta-Lapachone Nanotherapeutics for Lung Cancer Therapy(2009-01-08) Blanco, Elvin; Gao, JinmingA rising trend in lung cancer chemotherapy involves the development of agents that exploit molecular targets overexpressed in tumors, with hopes of increasing specificity, and in turn, efficacy. β-Lapachone (β-lap) is a novel anticancer drug whose mechanism of action relies on its bioactivation by the enzyme NAD(P)H:quinone oxidoreductase-1, NQO1, found overexpressed in NSCLC. While promising, its low water solubility limits its clinical translation. Moreovoer, a clinical formulation of the drug proves highly hemolytic and relatively ineffective. Our objective was to develop β-lapachone polymer micelles for lung cancer treatment, in hopes of targeting tumors: 1) pharmacokinetically, through the use of a platform that will ensure drug stability, bioavailability, and targeted localized delivery; and 2) pharmacodynamically, through the use of an anticancer drug that is bioactivated by a tumor-specific enzyme. We hypothesize that β-lapachone polymer micelles will result in a highly specific and effective nanotherapeutic platform for the treatment of lung cancer. Studies involving the clinical formulation of β-lap demonstrated that the vehicle, hydroxypropyl-β-cyclodextrin (HPβ-CD), causes hemolysis. The rapid dissociation of β-lap with HPβ-CD led to short blood circulation times and rapid distribution among all organs. By contrast, a micellar formulation of β-lap did not cause hemolysis, and displayed increased blood circulation times and relatively high and sustained accumulation in tumors. β-Lap micelles were small in size (~30 nm), possessed core-shell morphology, and displayed favorable release kinetics. In vitro examination of β-lap micelle efficacy in cancer cell lines demonstrated an NQO1-dependent mechanism of cell death. Upon translation to in vivo models of lung cancer in mice, β-lap micelles hindered tumor growth in subcutaneous lung tumors. It is important to note at this time that the clinical formulation of β-lap displayed no antitumor effects when compared to control tumors. Upon examination in an orthotopic model of lung cancer in mice, β-lap micelles were shown to prolong animal survival. Results from this study point toward the therapeutic potential of β-lap micelles for lung cancer treatment. Future studies involve optimization of the basic platform, as well as adjuvant use with current therapeutic strategies, to help propel this technology to preclinical studies and eventual clinical use.Item Characterization of mTOR Inhibition and Autophagy Inhibition in Non-Small Cell Lung Cancer(2014-04-14) Britt, Rebecca; DeBerardinis, Ralph J.; Minna, John D.; Levine, Beth; Mendelson, Carole R.Lung cancer continues to be the leading cause of cancer related death in both men and women. Pre-clinical studies of targeted therapies are needed in order to improve upon the chemotherapeutics that are currently in use. The ability to identify subsets of patient tumors which will respond to a particular targeted agent using biomarkers to indicate an acquired vulnerability will improve selection of effective therapeutics and minimize time and money wasted on ineffective drugs. The goal of this dissertation has been to characterize NSCLC response to mTOR inhibition and determine whether there are any molecular biomarkers that can predict response. mTOR is a central regulator of several pro-oncogenic signaling pathways and plays a role in cell growth, proliferation, metabolism, and inhibition of autophagy. Early studies examining mTOR inhibitors were limited by a lack of proper patient selection and the inability of first generation drugs to completely inhibit mTOR signaling. In the present study, we screened a panel of well-characterized NSCLC cell lines with three mTOR inhibitors, classical mTORC1 inhibitor rapamycin, and two novel dual mTORC1/2 inhibitors, Torin1 and AZD8055 in order to identify potential biomarkers that may be used to predict response to these agents. Additionally, in order to further characterize vulnerabilities to mTOR related genes within lung cancer subsets, we performed a genetic knockdown screen individually targeting 55 genes in this important pathway. Because inhibition of mTOR frequently leads to a cytostatic rather than cytotoxic effect, mTOR targeting agents may have greater utility when used in combination with other chemo- and targeted-agents. Therefore, we screened the three mTOR inhibitors in combination with the chemotherapy doublet paclitaxel/carboplatin or the targeted agent erlotinib. Finally, mTOR inhibition and other drug treatments have been shown to lead to autophagy activation. This process of cellular "self-eating" is thought to protect cancer cells from low nutrient availability and therapy induced stress. We screened NSCLC cells for their response to autophagy inhibitors alone and in combination with chemo- and targeted-therapy agents. The studies described in this thesis led us to the following conclusions. A subset of NSCLCs are more responsive to rapamycin than to mTORC1/2 inhibition by Torin1, or AZD8055, and sensitivity to mTOR inhibition is associated with RTK activation such as ERBB2 amplification or EGFR mutation or amplification, while KRAS and/or LKB1 mutations were associated with resistance. RNAi knockdown of various components related to mTOR signaling and autophagy produce a heterogeneous growth effect response in NSCLCs cells, and potentially define subset-specific vulnerabilities. mTOR inhibitors sensitize NSCLC cells to standard targeted- and chemotherapy agents erlotinib and paclitaxel/carboplatin doublet in an additive or synergistic manner, with the greatest level of synergy occurring in cell lines which are resistant to single agent therapies, including those with KRAS mutations. Finally, inhibition of autophagy using chloroquine is not likely to be a successful therapeutic approach in lung cancer as no significant growth effect was seen at physiologically relevant concentrations and no sensitization to standard chemo- or targeted-therapies were observed.Item Characterizing ASCL1-Dependent Neuroendocrine Non-Small Cell Lung Cancers(2013-06-27) Augustyn, Alexander; Amatruda, James F.; Minna, John D.; Johnson, Jane E.; White, Michael A.In order to achieve personalized medicine for the treatment of lung cancer, it is important to accurately classify tumors using a combination of factors, including patho-physiological features, tumor gene expression profiles, response to therapy, and oncogene/tumor suppressor mutation status. Gene expression analyses, including immunohistochemistry, single mRNA transcript analyses, and genome-wide mRNA expression profiling, performed over the course of the last three decades suggest that distinct, poorly performing neuroendocrine tumors occur in about 10% of otherwise pathologically indistinguishable non-small cell lung cancers. A complete molecular characterization of these tumors is lacking because no pre-clinical model exists. Utilizing genome-wide mRNA expression profiling from lung cancer cell lines established from a variety of patients, it was discovered that a rare subgroup of non-small cell lung cancer (NSCLC) lines demonstrated similar gene expression compared to a known neuroendocrine tumor, small cell lung cancer (SCLC). Validation of transcript analysis verified this data, and demonstrated that a particular transcription factor, ASCL1, required during development for the formation of pulmonary neuroendocrine cells, is dramatically upregulated in the subgroup of non-small cell lung cancer with neuroendocrine features (NE-NSCLC). Other cancer models have demonstrated addiction of tumors to developmental transcription factors and termed these genes “lineage oncogenes.” By showing that NE-NSCLC cell lines are addicted to ASCL1 expression and function, it was established that ASCL1 is also a lineage oncogene. Transcription factors of the basic helix-loop-helix category are historically difficult to target with small molecules, so a downstream target analysis was performed in order to understand the ASCL1 transcriptome. ChIP-Seq analysis demonstrated that ASCL1 regulates many genes, including several that are inherently druggable. Further studies proved that ASCL1 directly regulates the transcription of the anti-apoptotic regulator BCL2. Inhibition of BCL2 in vitro and in vivo led to induction of apoptosis and tumor xenograft regression suggesting that BCL2 is a potential therapeutic target in ASCL1-dependent NE-NSCLCs. Analysis of the upstream regulation of ASCL1 showed that it depends on a paradoxical activation of the RAS/RAF/MEK/ERK pathway. Small molecule agonists of this pathway were utilized to demonstrate reduction of ASCL1 levels and induction of apoptosis. The combination of ERK activation with BCL2 inhibition was then shown to be a viable therapeutic strategy for ASCL1-dependent tumors in vitro.Item Context-Selective Support of the AKT/mTOR Regulatory Axis by Tank-Binding Kinase 1 (TBK1)(2016-11-28) Cooper, Jonathan Mark; Brugarolas, James B.; Cobb, Melanie H.; Brekken, Rolf A.; White, Michael A.Oncogenic mutation of Ras or Ras effector signaling characterizes roughly thirty percent of all cancers. Persistent obstacles to the treatment of these diseases by direct Ras inhibition prompt alternative strategies aimed at leveraging signaling networks downstream of Ras. Tank-Binding Kinase 1 (TBK1) is downstream of the RalGEF/RalB arm of Ras effector signaling and supports Ras-driven oncogenic transformation via direct regulation of AKT. While TBK1 has been nominated as a therapeutic target, the field lacks knowledge of the mechanisms whereby TBK1 inhibitors mediate lethality and of the preferential context(s) for their application. We therefore leveraged toxicity profiles for TBK1 inhibitors in 100 NSCLC cell lines and identified robust correlation between TBK1 inhibitors and a cadre of mTOR direct and upstream regulatory network signaling inhibitors. This observation, along with orthogonal phosphoproteomics data, suggested an intersection exists between TBK1 and mTOR regulation and mechanistic target space. We identified that TBK1 is required for AKT/mTOR activation during the nutrient starved-to-fed state transition. Furthermore, we established that TBK1 physically intersects with the AKT/mTOR regulatory axis signaling at multiple nodes and can follow permissive and instructive mechanistic routes to regulate mTORC1 activation in response to nutrients. In parallel, we utilized a bioinformatics approach to identify that "Ras-mutant/mesenchymal" status serves as a molecular indicator of TBK1 inhibitor sensitivity in NSCLC. Concordantly, signaling through the AKT/mTOR regulatory axis was acutely attenuated by TBK1 inhibition in Ras-mutant/mesenchymal but remained unresponsive in Ras-mutant/epithelial NSCLC, indicating TBK1-resistant NSCLC may have uncoupled AKT/mTOR signaling from substantive TBK1 regulation. We furthermore demonstrated that TBK1 inhibition synergizes with Transforming Growth Factor-beta (TGF-beta)-mediated induction of the epithelial-to-mesenchymal transition (EMT) to reduce cancer cell viability. Together, these observations suggest that TBK1 supports pro-survival signaling downstream of Ras and EMT/TGF-beta signaling through the AKT/mTOR regulatory axis. Our findings, therefore, reveal novel mechanistic contributions of TBK1 in the regulation of AKT/mTOR signaling, and also nominate Ras-mutant/mesenchymal NSCLC as the preferential context for therapeutic interventions targeting TBK1.Item Defining Tumorigenic Contributions of Meiotic Cancer-Testis Antigens(2018-07-10) Nichols, Brandt Alan; Siegwart, Daniel J.; Cobb, Melanie H.; Westover, Kenneth D.; Whitehurst, Angelique WrightCancer Testis Antigens (CTAs) are expressed in testis and/or placenta and anomalously activated in a variety of tumors. However, the mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. A cohort of CTAs are required for recombination events during meiosis, suggesting meiotic CTAs have potential to functionally contribute to the genomic stability of tumors. To assess the tumorigenic contributions of meiotic CTAs, I employed a targeted siRNA screen for five meiotic CTAs. Depletion of SYCP1 or HORMAD1 decreased tumor cell proliferation, while SYCE1 loss resulted in elevated DNA damage. Another meiotic CTA, SPO11, is a topoisomerase that induces DNA double-strand breaks during meiosis. I found that SPO11 expression not only correlates with elevated DNA damage in a variety of tumor cells, but ectopic SPO11 expression increases DNA double-strand breaks. A chemigenomics approach identified that a meiotic CTA, HORMAD1, correlates with resistance to piericidin A in non-small cell lung cancer (NSCLC). Resistance is due to a reductive intracellular environment that attenuates the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibit elevated mutation burden and reduced survival. Differential expression profiling revealed that HORMAD1 tumors are enriched for genes essential for homologous recombination (HR). Mechanistic studies find that HORMAD1 promotes RAD51-filament formation but not DNA resection during HR. Accordingly, HORMAD1 loss enhances sensitivity to gamma-irradiation and PARP inhibition. Furthermore, HORMAD1 depletion significantly reduces tumor growth in vivo. These results suggest that HORMAD1 expression specifies a novel subtype of LUAD which has adapted to mitigate DNA damage. Altogether, these finding indicate that meiotic CTAs play functional roles in altering the genomic stability of tumors and represent potential intervention strategies to enhance sensitivity to DNA damage agents and/or immunotherapies in patients.Item Development of a Lung Cancer Targeting Peptide for Imaging and Drug Delivery(2018-04-12) Allred, Curtis Arlin; Corbin, Ian R.; Brown, Kathlynn C.; Kohler, Jennifer J.; Minna, John D.Lung cancer kills more people in the United Stated than the next three biggest cancer killers combined. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer patients and has an overall estimated 5-year survival of 18% in 2017. The emergence of targeted therapies (molecularly guided treatment) has improved the overall survival and quality of life of ~20% of NSCLC patients. A different molecularly guided treatment modality, targeting therapy, has shown success in other cancer types, but has not yet been successfully applied in NSCLC. Peptides are a class of molecules that have demonstrated exquisite targeting of cancer cells. HCC15.2 peptide was identified from a phage display library screen on NSCLC cells. It binds to an unknown receptor on a specific subset of NSCLC patient samples (24%) and cell lines (50%), which is not present in immortalized but non-transformed human bronchial epithelial cells (HBEC). Not only does HCC15.2 have a high affinity of ~5 nM and high specificity, it also triggers internalization and delivers its cargo into the cancer cell. HCC15.2 was optimized by altering the multimerization and amino acid content and protecting it from serum degradation. After the optimizations, HCC15.2 was shown to deliver many cargo types into cancer cells which traffic to and accumulate in lysosomes. HCC15.2 also demonstrated its ability to home to a subcutaneous xenograft in mice ~30-fold better than non-targeted dye after systemic delivery. This peptide used in molecular imaging could aid in earlier tumor detection, which correlates with better patient survival. HCC15.2 conjugated to saporin, a ribosome inactivating protein, showed an IC50 of 5.4 nM in in vitro viability assays. Delivery of saporin by HCC15.2 significantly slowed the growth of tumor xenografts. HCC15.2 is a perfect candidate for molecularly guided imaging and therapy.Item Discovery and Development of Predictive Biomarkers for the Personalization of Pemetrexed Therapy in Non-Small Cell Lung Cancer(2012-08-13) Watson, Misty Dawn Shields; Minna, John D.Lung cancer is a major health problem, and is the leading cause of cancer-related deaths. This research proposal was designed to personalize and improve overall response rates to the FDA-approved chemotherapeutic pemetrexed. To do so, clinically-applicable response phenotypes of a large panel of lung cancer lines needed to be defined to identify gene expression profiles driving response to pemetrexed treatment. To mimic clinical exposure and duration in vitro, published pharmacokinetics of patients treated with pemetrexed were extrapolated to develop the dose schedule used in this study. Although two drug response assays were simultaneously performed, liquid colony formation assay most closely mimicked clinical indications for the treatment of patients with pemetrexed, and was non-toxic to normal human bronchial epithelial cells. There were three distinct pemetrexed response phenotypes across this cell line panel: sensitive, intermediate and resistant. Interestingly, large cell carcinomas and a subset of adenocarcinomas are sensitive to pemetrexed treatment. Upon oncogenotype analysis, I found that mutations in EGFR correlated significantly with pemetrexed resistance, and this was confirmed by mRNA expression profiling and reverse phase protein arrays. Furthermore, I determined that mutations in KRAS were significantly more frequent in sensitive lines, and EML4/ALK adenocarcinomas conferred sensitivity to pemetrexed, consistent with recent clinical findings. In order to test whether the in vitro pemetrexed response phenotypes could be recapitulated in vivo, doses from 26.6 mg/kg up to 1000 mg/kg pemetrexed qwx3 were administered to and well tolerated by NOD/SCID mice. Treatment of established lung cancer xenografts with doses of pemetrexed 500 mg/kg or higher qwx3 were consistent with in vitro findings. To elucidate mechanisms of response, intratumoral mRNA levels of TYMS and pemetrexed-related genes did not have any significant correlation with response. However, microarray expression profiling, RNA-Seq and western blot analysis independently highlighted FTCD overexpression can be found only in pemetrexed-sensitive lung cancer lines. Stable knockdown of FTCD in a FTCD-overexpressed lung cancer line resulted in specific and abrupt cell death. I further describe the ongoing analysis of retrospective clinical datasets. Our plans for prospective FTCD biomarker enrollment in future pemetrexed clinical trials highlight the translational potential and progression of this dissertation proposal.Item EGFR mutations in non-small lung cancer: a basic science discovery with immediate clinical impact(2005-03-31) Dowell, Jonathan E.Item Elucidating the Target and Selectivity of a Non-Small Cell Lung Cancer Toxin(2019-07-11) Madhusudhan, Nikhil; Tu, Benjamin; Nijhawan, Deepak; DeBerardinis, Ralph J.; McFadden, David G.; Mishra, PrashantSelective toxicity among cancer cells of the same lineage is a hallmark of targeted therapies, and so identifying compounds that impair proliferation selectively is an important strategy in drug development. Here, we report the discovery of the quinazoline dione compound (QDC), a molecule that exhibits selective toxicity across 100 non-small cell lung cancer (NSCLC) lines. Using photoreactive probes, we found that the QDC targets mitochondrial Complex I. We investigated the selective toxicity of Complex I inhibition across NSCLC and found that a high baseline content of aspartate formed via reductive glutamine metabolism promotes resistance to mitochondrial inhibition. Altered metabolism is an important feature of cancer and there is significant interest in understanding how differences in tumor metabolism can be exploited for therapy. NSCLC cells subject to mitochondrial inhibition use reductive carboxylation to meet aspartate demand, which suggests that targeting reductive aspartate synthesis would enhance clinical response to Complex I inhibitors.Item Examining Lactate as a Fuel Source in Human Non-Small Cell Lung Cancer(2017-01-17) Doucette, Sarah; Faubert, Brandon; Cai, Ling; Hensley, Christopher T.; Yang, Chendong; Li, Hong; Huet, Giselle; Butt, Yasmeen; Torrealba, Jose; Oliver, Dwight; Ni, Min; Young, Jamey D.; Lenkinski, Robert E.; Kernstine, Kemp; DeBerardinis, Ralph J.Altered metabolism is a hallmark of cancer. Metabolic pathways such as glycolysis are enhanced in malignant tissue and can support the accelerated growth and proliferation of cancer cells. A longstanding dogma of cancer metabolism is that glucose is aerobically metabolized to lactate, as opposed to being oxidized in the TCA cycle. Additionally, lactate is thought to be excreted as a waste product by tumor cells. However, the metabolic behavior of cancer cells has been primarily studied in cultured cell systems rather than in bona fide tumors. Though useful to study the influence of intrinsic factors on metabolism, these systems do not account for extrinsic influences on metabolism that occur in vivo. In this work we study cancer metabolism in vivo through intra-operative nutrient infusions in patients with non-small cell lung cancer. Using 13C-stable isotope tracing, we found that glucose is highly oxidized in tumors compared to surrounding benign lung tissue. Next, we observed that lactate, in addition to glucose, can be used as a nutrient source by lung tumors. We examine the molecular mechanisms of this phenomenon in cell and animal systems using siRNA and CRISPR-mediated knockdown of the lactate transporters MCT1 and MCT4. These clinical observations highlight new aspects of in vivo cancer metabolism, which may lead to the development of new biomarkers or therapeutic opportunities.Item Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype(December 2021) Voth Park, Josiah Malachi; Kim, James; Minna, John D.; Brekken, Rolf A.; Akbay, Esra A.; Malter, James; Huang, LilyLung cancer is expected to kill ~150,000 people this year, encompassing 25% of all cancer related deaths making lung cancer the leading cause of cancer-related mortality in men and women. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) which represent 80-85% and 15-20% of cases, respectively. My dissertation project focused on understanding how to model the interactions between lung cancer cells, fibroblasts and immune cells. Immune cells are critical components of the tumor microenvironment (TME) that contribute to tumorigenesis, angiogenesis and metastasis. Macrophages are key regulators of the immune landscape within the TME. The plasticity of macrophage phenotypes in the TME correlates with prognosis of NSCLC. Depending on their phenotype, macrophages in the TME can secrete pro-tumor cytokines and chemokines, ultimately suppressing the function of anti-tumor immune cells in the TME. The purpose of my project was to investigate if and how NSCLC cells alter macrophage phenotype in multi-cellular co-cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. The central hypothesis of the project is, tumor cell characteristics drive macrophage polarization in the TME, and this can be captured using a multicellular co-culture model. Given the central importance of macrophages to the TME and the immune landscape of NSCLC, an understanding of the tumor cell characteristics associated with immune suppressive or immune stimulatory macrophage phenotype could be exploited from a therapy perspective in the future. To address this hypothesis, an in vitro co-culture system (NSCLC tumor cells, human cancer associated fibroblasts (CAFs), and mouse macrophages) was developed to interrogate cancer cell features driving heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured: mRNA expression in mouse macrophages with a panel of qPCR probes for genes associated with distinct macrophage phenotypes (Arg1, iNOS, Il-1β, Il-6, Ym-1, Socs3). This system was validated by comparison of macrophage phenotypes represented in the TME of lung cancer xenografts grown in athymic nude mice. Using our platform, we evaluated ~80 NSCLC patient derived lines for their effect on mouse macrophage phenotype. We identified three main macrophage phenotypes across this panel of NSCLCs. To identify cancer cell biomarkers for macrophage polarization, we interrogated molecular characteristics of the cancer lines. Additionally, we expanded the functionality of the platform to assess the effects of pharmacologic agents on macrophage phenotype. As a proof of principle, a small panel of known immune stimulating compounds was tested in the in vitro co-culture platform and validated in human tumor xenografts. Finally, we identified a few novel compounds that show selective cancer cell toxicity and reprogram macrophage phenotype. In conclusion, we built a reproducible in vitro platform to interrogate macrophage polarization in the TME. We leveraged this platform to identify three dominant macrophage phenotypes induced by NSCLC cells and CAFs. We found that no cancer cell molecular characteristic alone drives macrophage polarization. Finally, we illustrate the significance of this platform for immune stimulating drug identification; we identified two novel chemicals that repolarize macrophages and kill cancer cells simultaneously.Item Exploration of Nuclear Receptor Activity and siRNA-Derived Phenotypes as Therapeutics in Non-Small Cell Lung Cancer(2015-04-10) Carstens, Ryan Murray; Kittler, Ralf; Minna, John D.; Mangelsdorf, David J.; Kliewer, Steven A.; White, Michael A.Nuclear hormone receptors are master regulators of diverse cellular functions implicated in tumor pathogenesis and as oncogenic drivers of many human cancers. To better understand what role these important receptors might be playing in lung cancer, three interconnected studies were initiated to assess nuclear receptor function, expression, and drugability within the lung cancer context. First, a "technology development" project was undertaken to produce and troubleshoot a CLIA-certifiable, high-throughput biomarker platform capable of mRNA expression signature assessment from FFPE specimens. The platform was used to assess NR/CoReg expression levels across a 500+ sample FFPE dataset. Categorical NR/CoReg downregulation upon tumor progression as well as survival benefits for patients retaining a non-pathological NR/CoReg expression pattern were discovered. Second, a panel of 110 NR ligands was screened across a 100-member cell line panel representative of all clinically-relevant facets of lung and breast cancers to pharmacologically interrogate these receptors as novel lung cancer targets. Following completion of this screening effort, three classes of ligands targeting the estrogen, glucocorticoid, and vitamin D receptors (ER, GR, and VDR respectively) that exert anti-proliferative phenotypes on specific subsets of the lung cancer cell lines were identified. Of particular note, several of these agents are routinely used in current clinical practice (particularly dexamethasone) and represent excellent candidates for rapid clinical translation of these findings. Finally, an RNAi-based systematic functional interrogation of NR/CoReg function was undertaken in a 100+ member cell line panel representative of all clinically-relevant facets of lung and breast cancers. A reproducible classification of lung and breast cancers was defined based on their holistic functional states as represented by the RNAi dataset. Each of these "clades" of cancer cell lines was demonstrated to be specifically targetable by unique siRNA reagents capable of inducing growth attenuation or amplification in only that clade of cell lines. Further investigation into the mechanisms of action of these siRNA reagents unexpectedly revealed that the phenotypes were largely mediated by miRNA-like seed sequence based effects rather than target-directed siRNA total complementarity silencing. Following this discovery, efforts were undertaken and subsequently completed to identify the "true" targets of these clade-specific siRNAs.Item Heterogeneity in Human NSCLC Tumor Glucose Metabolism: On the Origins and Role of Pyruvate Metabolism(2015-07-22) Hensley, Christopher Thomas; Zhu, Hao; DeBerardinis, Ralph J.; Tu, Benjamin; Shay, Jerry W.In 1956 Otto Warburg, the most prominent scientist in cancer metabolism, stated that "the problem of cancer is...to discover the differences between cancer cells and normal growing cells" (Warburg, 1956a). Over fifty years later, the field still lacks a valid experimental framework to discover such metabolic differences in human tumors. A major limitation is the inability to faithfully recapitulate the microenvironment of primary human tumors in model systems. As a result, fundamental questions about tumor metabolism, including the suppression of pyruvate oxidation upon transformation proposed by Warburg more than 50 years ago, have only rarely been subjected to direct experimental assessment. To provide a direct readout of primary human tumor metabolism in vivo, we have used intra-operative 13C-glucose infusions in non-small cell lung cancer (NSCLC) patients to compare metabolism between tumors and non-cancerous lung. Pre-surgical imaging, including non-invasive assessment of tissue perfusion using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), allowed us to select areas of microenvironment-based heterogeneity, to guide sample acquisition. Specifically, this microenvironment-based heterogeneity was assessed relative to the oncogenotype, histological parameters, and metabolism of glucose through glycolysis and the TCA cycle. Diverse tumors displayed enhanced glycolysis and glucose oxidation. Furthermore, we discovered that due to the low enrichment in acetyl-CoA and other TCA cycle intermediates, all tumors had evidence for oxidation of multiple nutrients. We identified lactate as a carbon source for tumor oxidative metabolism. Additionally, metabolically heterogeneous regions were identified within and between tumors using DCE-MRI. Regions of lesser contrast enhancement demonstrated higher 13C enrichment, likely reflecting contributions of non-glucose nutrients to central carbon metabolism in well-perfused areas, or the cause or consequence of aberrant proliferation of aggressive clones resulting in inadequate perfusion. The data indicate that the heterogeneous metabolism of these tumors is highly and predictably related to the microenvironment. In summary, we have made novel, significant progress in assaying and analyzing primary human tumor metabolism and its relation to the microenvironment in vivo. I close with a separate project for future directions to begin to dissect the cellular origins of the whole tumor fragment signal that is amenable to direct assays in patients.Item Identification and Characterization of Non Small Cell Lung Cancer Stem Cells(2011-02-01) Sullivan, James Patrick; Minna, John D.The discovery of rare tumor cells with stem cell features first in myeloproliferative disease and later in solid tumors has emerged as an important area in cancer research. Through these studies the cancer stem cell model has emerged, which postulates that many tumors are initiated and progressed by a population of self-renewing malignant stem cells, referred to as cancer stem cells. This new tumor growth paradigm suggests that tumor metastasis and recurrence may be driven by a residual population of highly aggressive cancer stem cells. Furthermore this model argues that complete cancer remission may only be achieved by eradicating the malignant stem cell population charged as the source of tumor cell renewal. Lung cancer is the most commonly lethal form of cancer in the world with about 90% of the nearly one million new cases succumbing to the disease. While progress is being made in understanding lung cancer pathogenesis and improving therapy, prognosis remains poor. One approach to improving outcome in lung cancer has been to therapeutically target a unique, phenotypically defined lung cancer stem cell population. However despite the relatively rapid pace of cancer stem cell research in solid tumors such as breast, brain and colon cancers, similar progress in lung cancer remains hampered in part due to an incomplete understanding of lung stem cell hierarchy and the complex heterogeneity of the disease. To address this challenge, putative lung cancer stem cells were prospectively isolated from patient lung tumors and lung tumor cell lines using methods that have been reported to enrich for other stem cell populations in other cancers. As a result, a subpopulation of cells with elevated aldehyde dehydrogenase (ALDH) activity within many NSCLCs was identified with properties indicative of a cancer stem cell population including enhanced tumorigenicity in xenograft models, clonogenicity in culture and the capacity for self-renewal. In support of this, analysis of 282 clinically annotated non small cell lung cancer samples found elevated ALDH1A1 expression, the protein that drives ALDH in lung cancer, was associated with poor patient prognosis. Finally, molecular characterization of isolated ALDH+ lung cancer cells revealed elevated expression of stem cell transcripts including Notch signaling transcripts, suggesting enhanced pathway activity. Suppression of Notch signaling through chemical inhibition or knockdown of the proto-oncogene NOTCH3 resulted in a significant reduction in clonogenic ALDH+ cells indicating the importance of Notch signaling in lung cancer stem cell homeostasis and as a potential target for lung cancer stem cell directed therapy.Item Identification of Biomarker Driven Intervention Opportunities and Advancement of Mechanism of Action Predictions for Anti-Cancer Therapeutics(2017-04-14) McMillan, Elizabeth Anne; Cobb, Melanie H.; White, Michael A.; Ranganathan, Rama; Minna, John D.Oncogenic lesions arising during cancer progression provide an attractive target for chemical intervention strategies. The extreme molecular heterogeneity of tumors, however, makes it difficult to identify authentic intervention targets and to link patients to the most appropriate treatment. To confront this challenge, we launched a full scale investigation to identify the genetic lesions that arise during cancer progression together with a computational approach to link novel compounds to these lesions. A panel of 103 non-small cell lung cancer cell lines was screened with over 200,000 uncharacterized synthetic chemical compounds and natural products fractions in a tiered HTS approach. Statistical and machine learning procedures were then used to link drug activity to the complexity of cancer genomes by systematically assigning enrollment biomarkers to each compound from measures of gene expression, gene mutation, gene copy number, protein expression, and metabolomics datasets. Using this approach, we have found that genetic vulnerabilities that are not currently actionable can be linked to novel chemicals. Experimental mechanism of action hypotheses can be derived from these chemical/biomarker relationships and were validated for a subset. Notably, we are able to parse KRAS mutant cancers into multiple, distinct molecular subtypes defined by co-occurring mutations. This indicates that KRAS lung cancers are representative of diverse mechanistic subtypes, and we are able to identify putative novel compounds that may target each subtype. Collectively, we are using this approach as a data driven way to parse mechanistic cancer subtypes and identify a diverse cohort of therapies capable of contending with cancer heterogeneity together with enrollment biomarkers that can specify sensitivity.Item Identification of Drivers of Tumor Lymphangiogenesis in Non-Small Cell Lung Carcinoma (NSCLC)(2014-02-04) Sibley, Robert Carson; Dellinger, Michael; Brekken, RolfBACKGROUND: Non-small Cell Lung Carcinomas (NSCLCs) frequently spread to regional lymph nodes before they colonize other regions of the body, and the status of regional lymph nodes is an important prognostic factor for predicting the outcome of patients with lung cancer. It has recently been demonstrated that lymphangiogenesis, the sprouting of new lymphatic vessels from pre-existing vessels, facilitates the lymphogenous dissemination of NSCLC. However, the molecular mechanisms driving lymphangiogenesis in NSCLC are poorly understood. Objective: Our aim was to identify novel lymphangiogenic genes by identifying lymphangiogenic lung tumor cell lines, and then to use microarray data to generate a "lymphangiogenic" gene signature. METHODS: Tumors from 13 lung tumor cell lines were stained with antibodies against LYVE-1 and Podoplanin. Lymphatic vessels were counted in 5 representative 20X fields per tumor. Average lymphatic vessel densities were then calculated. Cell lines were grouped into lymphangiogenic, non-lymphangiogenic, and intermediate categories. Microarray data from the two extreme groups were then compared to generate a "lymphangiogenic" signature. RESULTS: Four cell lines, (Calu-1, H1993, HCC461, and HCC827) displayed high intratumoral lymphatic density, and five cell lines (Calu-3, H1155, H1395, H1975, and H2073) displayed no intratumoral lymphatic vessels. The "lymphangiogenic" signature obtained from the microarray data from these groups contained 146 genes, including the lymphatic growth factor VEGF-C. CONCLUSIONS: Our preliminary findings suggest that VEGF-C is an important driver of tumor lymphangiogenesis in NSCLC. The other 145 genes in the signature may also serve novel functions in regulating tumor lymphangiogenesis. Together, the results from this project provide mechanistic insight into the process of tumor lymphangiogenesis and metastasis. We believe that this information will lead to the development of new prognostic or predictive markers and therapeutic strategies to improve the outcome of patients with lung cancer.