Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expression. In Paper IV, the diagnostic methods for identification of ALK rearrangements were assessed in a large representative Swedish NSCLC population. Fluorescence in situ hybridization (FISH), as the diagnostic standard, was compared to two immunohistochemical assays. ALK gene expression levels were incorporated to supplement the molecular data. The frequency of ALK rearrangements was lower than previously reported. The different methods to detect the ALK fusion demonstrated overlapping results. However, the overlap was poor, so the methods cannot be regarded as interchangeable and should thereby be interpreted with caution when used in clinical diagnostics. In summary, this thesis applied an integrative translational approach to characterize potential new treatment targets and to evaluate the detection of existing predictive biomarkers in NSCLC.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Cancer is a group of diseases that are characterized by uncontrolled growth and spread of cells. In order to treat cancer successfully, it is important to diagnose cancers in their early stages, because survival often depends on the stage of cancer detection. For that purpose, highly sensitive and selective methods must be developed, taking advantage of suitable biomarkers. The expression levels of proteases differ from one cancer type to the other, because different cancers arise from different cell types. According to the literature, there are significant differences between the protease expression levels of cancer patients and healthy people, because solid tumors rely on proteases for survival, angiogenesis and metastasis. Development of fluorescence-based nanobiosensors for the early detection of pancreatic cancer and non-small-cell lung cancer is discussed in this thesis. The nanobiosensors are capable of detecting protease/arginase activities in serum samples over a broad range. The functionality of the nanobiosensor is based on Förster resonance energy transfer and surface energy transfer mechanisms. The nanobiosensors for protease detection feature dopamine-coated Fe/Fe₃O₄ nanoparticles, consensus (cleavage) peptide sequences, meso-tetra(4-carboxyphenyl)porphine (TCPP), and cyanine 5.5. The consensus peptide sequences were synthesized by solid-supported peptide synthesis. In this thesis, improved consensus sequences were used, which permit faster synthesis and higher signal intensities. TCPP, which is the fluorophore of the nanoplatform, was connected to the N-terminal end of the oligopeptides while it was still on the resin. After the addition of TCPP, the TCPP-oligopeptide was cleaved off the resin and linked to the primary amine groups of Fe/Fe₃O₄-bound via a stable amide bond. In the presence of a particular protease, the consensus sequences attached to the nanoparticle can be cleaved and release TCPP to the aqueous medium. Upon releasing the dye, the emission intensity increases significantly and can be detected by fluorescence spectroscopy or, similarly, by using a fluorescence plate reader. In sensing of arginase, posttranslational modification of the peptide sequence will occur, transforming arginine to ornithine. This changes the conformational dynamics of the oligopeptide tether, leading to the increase of the TCPP signal. This is a highly selective technology, which has a very low limit of detection (LOD) of 1 x 10⁻¹⁶ molL⁻¹ for proteases and arginase. The potential of this nanobiosensor technology to detect early pancreatic and lung cancer was demonstrated by using serum samples, which were collected from patients who have been diagnosed with pancreatic cancer and non-small cell lung cancer at the South Eastern Nebraska Cancer Center (lung cancer) and the University of Kansas Cancer Center (pancreatic cancer). As controls, serum samples collected from healthy volunteers were analyzed. In pancreatic cancer detection, the protease/arginase signature for the detection of pancreatic adenocarcinomas in serum was identified. It comprises arginase, MMPs -1, - 3, and -9, cathepsins -B and -E, urokinase plasminogen activator, and neutrophil elastase. For lung cancer detection, the specificity and sensitivity of the nanobiosensors permit the accurate measurements of the activities of nine signature proteases in serum samples. Cathepsin -L and MMPs-1, -3, and -7 permit detecting non-small-cell lung-cancer at stage 1.

Pemetrexed (PTX), an antifolate drug, has been approved by the US FDA for first line therapy of mesothelioma and non-small cell lung cancer. In addition to its primary site of action on thymidylate synthase (TS), PTX also inhibits the second folate-dependent enzyme of purine biosynthesis aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The accumulation of the substrate for AICART, ZMP, in PTX-inhibited cancer cells leads to activation of AMP-activated protein kinase (AMPK) with subsequent inhibition of mammalian target of rapamycin (mTOR) and hypophosphorylation of its downstream targets responsible for protein synthesis and cell proliferation. Inhibitors of mTORC1 like Rapamycin and its analogs (rapalogs) have only partial effects on tumor cells as they do not inhibit mTORC2, which phosphorylates Akt subsequently relieving the inhibition of mTORC1, thus leading to poor cytotoxicity by rapalogs. AMPK exerts control on mTORC1 kinase activity and PTX mediated activation of AMPK leads to its subsequent downregulation and hence, would be expected to have a therapeutic interaction with direct mTOR inhibitors. AZD8055, an ATP-competitive inhibitor of mTOR kinase, potently inhibits both mTORC1 and mTORC2 and therefore, can overcome the feedback mechanism(s) limiting the action of rapalogs to cytostatic effects. To study the effects of AMPK activation and mTOR inhibition pharmacologically, we performed growth suppression assays using pemetrexed, AICAR, RAD001, and AZD8055. The effect of inhibition of mTOR with these drugs was assessed by examining the dephosphorylation of mTORC1 substrates S6K1 and 4E-BP1, as single agents and in combination, at their 50% inhibitory concentrations (IC50) by western blotting. Our data suggested that AMPK activation via PTX mediated AICART inhibition in combination with direct mTOR inhibition by AZD8055 has a synergistic interaction on the proliferation of NSCLC cells in culture. Inhibition of mTOR endogenously by pemetrexed, along with direct pharmacological inhibition of mTOR prevents the feedback circuit which may compromise the therapeutic efficacy of rapamycin analogs. Pemetrexed and AZD8055, as single agents, demonstrated inhibitory activity on phosphorylation events of mTORC1 substrates. This activity was markedly increased by combining both the drugs. Our findings suggest that direct inhibitors of mTOR enhance the effects of activators of AMPK. These effects appear to be mediated via combined effects on mTORC1. Taken together, the combination of catalytic site mTOR inhibitors and pemetrexed is a promising therapeutic strategy and calls for further preclinical and clinical investigations.

No description available.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

Background: Randomized controlled trials (RCTs) are the gold standard for assessing the efficacy of a medical treatment. However, the efficacy demonstrated by trials does not automatically translate into a comparable level of effectiveness in the real world. RCTs may vary from routine clinical practice in several ways; the patients themselves, the delivery of the treatment, and the collateral care provided during treatment. Phase IV studies that assess outcomes of a treatment in the real-world provide a mechanism for assessing treatment effectiveness. Objectives: The objectives of this study were to: describe the characteristics of patients receiving standard, first-line, palliative, platinum-doublet chemotherapy (PPDC) for non-small cell lung cancer (NSCLC) in routine care; describe the effectiveness of PPDC in terms of wellbeing and symptom control; identify patient characteristics associated with change in wellbeing with treatment; and compare reported treatment efficacy to the effectiveness observed in the current study. Methods: This study was a retrospective cohort study of patients treated at Ontario’s Regional Cancer Centres (RCCs). Patients’ Edmonton Symptom Assessment System (ESAS) scores were used to describe patients’ symptomatic status and wellbeing. The proportions of patients whose wellbeing improved, remained stable or deteriorated at two months were calculated. Using logistic regression, patient and disease characteristics were assessed for association with change in wellbeing at two months (dichotomized as improved/stable and deteriorated). In comparing trial results to this study, adjustments were made for differences in case mix. Results: Patients’ median age was 65, 55% were male and the majority had stage IV disease and adenocarcinoma histology. Patients’ baseline wellbeing and symptomatic status varied widely. 61.3% (95% CI: 55.8 – 66.6%) of patients had improved or stable wellbeing at two months. Histology and baseline wellbeing score were associated with change in wellbeing at two months. The case mix adjusted estimates of the proportion of improved/stable patients (60.0% (95% CI 54.5 – 65.3) and 60.5% (95% CI 54.9 – 65.6)) were consistent with the proportion of patients achieving general quality of life improvement or stabilization in RCTs (55% and 63%). Conclusion: The effectiveness of PPDC delivered in Ontario’s RCCs is consistent with that expected based on the results of RCTs. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-04-23 11:53:33.491

Lung cancer is the most common cancer worldwide and is the fourth leading cause of death in South Africa. Lung cancer is categorised into two types; non-small cell lung cancer and small cell lung cancer (SCLC). SCLC constitutes 20% of all lung cancers and is considered to be an aggressive tumour as it gains chemo-resistance and exhibits early metastasis in diagnosed patients. SCLC cells originate from the neuroendocrine cells of the bronchoepithelium and are known to secrete the neuropeptide, proopiomelanocortin (POMC). POMC undergoes proteolytic cleavage to produce the adrenocorticotropin hormone (ACTH). ACTH stimulates the production of the steroid hormone, glucocorticoid hormone (GC), through the hypothalamus-pituitary-adrenal (HPA) axis. The produced GCs mediate a negative feedback system of the HPA axis to sequester ACTH production. SCLC cells are insensitive to this negative feedback stimulus. GCs elicit their actions through the glucocorticoid receptor (GR). Studies have shown that SCLC cells have a reduced expression of GR which perpetuates the GC-insensitivity. Importantly, over-expression of exogenous GR in SCLC cells leads to cell death by apoptosis. It was postulated that SCLC cells select against GR expression for longevity. Cancer cells are known to alter/silence the expression of tumour suppressor genes by a mechanism known as methylation. Methylation occurs when the enzyme, DNA methyltransferase 1, adds a methyl group to a cytosine present in a guanine-cytosine rich region of the gene (CpG island). The GR gene has a 5’-untranslated exon 1 region that consists of eight promoter regions (1A-1J), in these promoter regions are many CpG islands that have the potential to be methylated. The first aim of this study was to determine the promoter/s utilised by SCLC cells to express the GR protein. Conventional PCR revealed that all three cell lines predominantly utilise promoters 1B and 1C for GR expression. Bioinformatic analysis revealed that these promoters contain putative CpG islands and new data suggests that the GR is silenced by methylation and that treatment with a de-methylating agent results in GR re-expression. To determine which promoter is responsible for GR re-expression after de-methylation, the SCLC cell line, DMS79, as well as two control cell lines, A549 and HEK cells, were treated with the de-methylating agent, 5-aza-2’-deoxycytidine, for 72 hours. qPCR analyses revealed that all three cell lines expressed promoters 1B and 1C with A549 cells showing no evidence of methylation. The HEK cells showed methylation in promoter 1C and not promoter 1B. The SCLC cells showed methylation in both promoter 1B and 1C, however, only promoter 1B showed a significantincrease in transcript levels. SCLC cells are induced to undergo GC-mediated apoptosis when GR expression is restored however the mechanism utilised by the GR to induce the apoptotic cascade is unknown. The GR structure is divided into three domains; ligand binding domain (LBD), DNA binding domain (DBD) and amino terminal domain (NTD). The second aim of this study was to determine the component of the GR that induces apoptosis of SCLC cells. HEK and SCLC cells were infected with empty virus and various GR construct viruses; containing either a wild-type GR, ligand binding mutant, DNA binding mutant or a transactivation mutant (NTD); for 72 hours. Both cell lines were quantified for apoptosis and cell death using microscopic analyses. In HEK cells, it was shown that apoptosis occurred in cells expressing the wild-type GR, the DNA binding mutant and transactivation mutant constructs but apoptosis was reduced in cells expressing the ligand binding viruses. This indicates that the LBD may be necessary for inducing apoptosis in HEK cells. In DMS79 cells, apoptosis occurred in cells expressing the wild-type GR, ligand binding mutant and the DNA binding mutant constructs. There was less apoptotic activity exhibited in the transactivation constructs which indicates the NTD may be necessary for apoptosis induction in these cells. The NTD of the GR is responsible for interaction with other transcription factors to mediate GR transcriptional activity and this study has shown that the transactivation domain plays a necessary role in apoptosis induction. An analysis of the various pathways the GR interacts with through the NTD domain could lead to the identification of the pathway which triggers apoptosis in SCLC cells. This discovery, together with knowledge of promoter methylation and expression may contribute to the development of new, more effective therapies for SCLC. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Lung cancer is the leading cause of cancer-related deaths among men and women. Non-Small Cell Lung Cancer (NSCLC) constitutes the most common type of lung cancer and is frequently diagnosed at advanced stages. In the past decade, discovery of Epidermal Growth Factor Receptor (EGFR) mutations have heralded a new paradigm of personalized treatment for NSCLC. Clinical studies have shown that molecular targeted therapies increase survival and improve quality of life in patients. Despite these advances, the realization of personalized therapies for NSCLC faces a number of challenges including the integration of clinical and genetic data and a lack of clinical decision support tools to assist physicians with patient selection. This thesis demonstrates the development of a predictive computational model for personalized therapeutic interventions in advanced NSCLC. The findings suggest that the combination of clinical and genetic data significantly improves the model’s predictive performance for tumor response than clinical data alone.

Multidrug resistance (MDR) in small cell lung cancer is one of the major causes of failures of chemotherapy. MDR is a means of protection of tumor cells against chemotherapeutic drugs. Although the molecular basis of MDR is not fully understood, genes involved in apoptosis may be mutated. Recent finding of a link between over-expression of an apoptotic gene, cyclooxygenase 1 (cox 1), and MDR suggests that cox 1 is involved in the development of MDR phenotype. This research was an attempt to observe whether up-regulation of cox 1 contributes to the MDR phenotype in small cell lung cancer cells. This research ultimately may provide a mechanism to reverse the abberant up-regulation of apoptosis genes associated with multidrug resistance to either eliminate or control reproduction of cancer cells. Real time RT PCR was used to confirm the up-regulation of cox 1 in cultured MDR resistant small cell lung cancer cells (GLC4). The up-regulated cox 1 expression was down-regulated using RNA interference technology (RNAi) by transfection with an anti-cox 1 siRNA. More than 90% transfection of cells was confirmed using confocal microscopy. Down-regulation of cox 1 was validated as the protein expression significantly decreased (P=0.004) from multidrug resistant small cell lung cancer transfected cells compared to multidrug resistant nontransfected cells. There was decrease level of expression of cox 1 in multidrug resistant cells after the knockdown with siRNA specific to cox 1. The decreased level of cox 1 expression and, therefore, Cox 1 production increased the rate of apoptosis in small cell lung cancer cells as indicated by its sensitivity to the doxorubicin. / Department of Biology