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Cancer is among the most common causes of human deaths globally. Because of limitations and adverse effects of conventional cancer treatments, the need for new treatments is imminent. A rapidly expanding field in cancer therapy is cancer immunotherapy, which aims to, in one way or another, aid the patient’s own immune system in its battle against the tumor cells. A type of cancer immunotherapy is oncolytic virotherapy which utilizes viruses that either have a natural inclination to infect and replicate inside tumor cells or have been engineered to specially replicate in tumor cells causing oncolysis. An example of an oncolytic virus is the Lokon Oncolytic Adenovirus (LOAd). This virus specifically replicates inside cancer cells and is based on adenovirus serotype 5 but with a serotype 35 fiber, causing it to infect via the cluster of differentiation 46 receptor, which is ubiquitously expressed on somatic and tumor cells. A notable virus with the LOAd backbone, that is being evaluated in several clinical trials, is LOAd703, which is armed with the immunostimulatory transgenes 4-1BB ligand and a trimerized cluster of differentiation 40 ligands. In this project, I describe the development and evaluation of Ad703+, a cell killing adenovirus carrying the transgenes of LOAd703 as well as a novel cytotoxic transgene that never has been used in oncolytic virotherapy previously. This virus was developed using the AdEasy system, which is a replication-deficient virus platform based on adenovirus serotype 5. This virus enters cells using the human coxsackie and adenovirus receptor, which is homologous to the murine equivalent.  The ability to express the immunostimulatory transgenes and the cell killing ability of Ad703+ was evaluated in two different human cell lines, HEK293 which allows the replication of Ad703+, and the lung cancer model A549. Ad703+ was shown to express the immunostimulatory transgenes in both of the cell lines, but in a replication-dependent manner. Ad703+ was also shown to exhibit cell killing ability in a replication-independent manner on par with other oncolytic viruses.  The ability of Ad703+ to trigger cell death in a replication-independent manner opens up for the possible application in pre-clinical in vivo studies using mice due to its theoretical ability to infect murine cells and simulate viral oncolysis.

Micro ribonucleic acids (miRNAs) are pivotal post-transcriptional regulators of gene expression and if research continues to reveal positive results, they will soon be used as therapeutic targets in the clinical setting for the treatment of a variety of cancers. They are evolutionarily conserved small noncoding RNAs that range from 18 to 24 nucleotides in length. There are over 1,400 miRNAs for which abundant evidence has demonstrated fundamental importance in normal cell development and up- or downregulation when they become deregulated. The deregulation of miRNAs, which can function as tumor suppressors or oncogenes, contributes to the development of cancer, among other diseases. Deregulation of tumor suppressor miRNA can occur in many different tissues of the body and lead to a variety of cancers. Tumor suppressor let-7 negatively regulates expression of an oncogenic mRNA named RAS. In lung cancer, a decrease in let-7 produces an increase in expression of RAS, which contributes to cell proliferation and tumorigenesis. In chronic lymphocytic leukemia, Bcl2 protein becomes overexpressed due to the down-regulation of tumor suppressors miRNA-15 and miRNA-16. MicroRNA-34 plays an important role in neuroblastoma in which its expression is decreased due to mutations that decrease a tumor suppressor protein, p53. Upon deregulation of oncogenic miRNAs, tumor suppressor mRNA expression is decreased and leads to multiple types of cancer. Up-regulation of the miRNA-17-92 cluster in lung cancer leads to increased cell proliferation and contributes to angiogenesis in many cancers. In B cell lymphoma, miRNA-155 becomes up-regulated along with an RNA named BIC. This up-regulation accelerates pathogenesis and up-regulation of an oncogenic protein, c-myc. MicroRNA-21 acts as an anti-apoptotic factor by downregulating apoptosis-related genes and contributing to the development of human glioblastoma. This review summarizes the present understanding of how miRNAs function at the molecular level in the body, how their deregulation contributes to tumor formation, maintenance and metastasis and how they can be used for cancer diagnosis, prognosis and therapy. With the mass amounts of knowledge gained from the current research done on miRNAs, a cancer cure may soon be developed based on the targeting of specific miRNAs. The promise of miRNAs in cancer therapeutics will depend on the development of proper delivery strategies of miRNA mimics and inhibitors, in addition to evaluation of safe usage and toxicity of therapeutic dosages.

Glioblastoma is the most malignant form of gliomas and is a highly infiltrative while non-metastatic tumor of the central nervous system. Patients with glioblastoma have a poor prognosis of 15 months median survival after diagnosis. Promising results were reported in recent clinical trial regarding glioblastoma treatment with chimeric antigen receptor (CAR) T therapy. The lab has previously developed five novel scFvs targeting IL13R⍺2, a tumor-associated antigen in glioblastoma, and integrated them into the second-generation CAR. We named them, 10CAR, 27CAR, 55CAR, 75CAR and 117CAR. The ex vivo cytotoxicity and proliferation assay demonstrated that the 117CAR T construct has the best functionality, while 27CAR T construct has a poor functionality compared to the rest of the constructs. FACS analysis was performed to check the CAR expression in different constructs. 27CAR T cells showed the lowest surface CAR expression and 117CAR T cells displayed the highest out of five constructs. 27CAR T cells were also activated more without stimulation compared to other constructs. We selected out 27CAR and 117CAR T cells for the further investigation to understand the attribution of the discrepancy between 27CAR and 117CAR T cells.   We observed a larger cellular size for 27CAR T cells compared to the rest constructs in flowcytometry analysis, which is usually associated with activation. IFN-γ production of all constructs without target cells stimulation were detected to examine the activation state of different constructs. We observed the highest IFN-γ production in 27CAR T cells without stimulation. These results together indicate that a potent antigen-independent activation or, in other words, tonic signaling is present in 27CAR T cell. The tonic signaling further leads to an early exhaustive phenotype of 27CAR T cells, that is not present in 117CAR T cells. Removing the endodomain of CAR rescued the antigen-independent activation and early exhaustion of 27CAR T cells. The surface and total CAR expression of 27CAR and 117CAR T cells were determined by flowcytometry. 27CAR T cells presented a lower expression of both surface and total CAR. A significantly lower percentage of total CAR on the surface indicates the internalization of CARs in 27CAR T cells. Removing the intracellular domain of 27CAR did not restore the surface expression of CAR. 27CAR and 117CAR differ in four CDRs of scFv, CDR1, 2,3 in the heavy chain and CDR3 in the light chain. We replaced all the amino acids differing between these two constructs with alanine in a CDR-by-CDR manner and obtained five alanine substitution constructs. We then analyzed the CAR expression in Jurkat cells, and we found that the trafficking of CAR to the surface was significantly improved by mutating the CDR2 in the heavy chain or CDR3 in the light chain. Moreover, when the two CDRs were replaced simultaneously, almost all transduced cells expressed CAR, as was the case of cells transduced with 117CAR.   To summarize, the tonic signaling induced by higher tendency of clustering of 27scFv results in the antigen-independent activation and early exhaustion of 27CAR T cells. By removing the endodomain of 27CAR, we abrogated the phenomenon. Further, CDR2 in heavy and CDR3 in light chain in 27scFv are responsible for the impaired trafficking of CAR to the surface.

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Aim: Several studies have tried to quantify overdiagnosis of prostate cancer with Prostate-specific antigen(PSA) screening, but estimates vary widely. This study aims to evaluate the degree of overdiagnosis of prostate cancer with 10 or 14 follow-up years after the stop of screening in Finland. Methods: We selected 80379 men aged 55-69 years who were randomized to a screening or a control arm, distinguishing four birth cohorts: 1941-44,1937-40, 1933-36 and 1929-32. The first PSA screening test occurred during1996-1999. Men without detected as prostate cancer in the previous screening would be invited to the next screening 4 years later. The estimate of overdiagnosis is the ratio of the cumulative excess incidence to the cumulative incidence of prostate cancer in the screened group after the year-specific incidence became stable. Results: The patterns of all incidences in these four cohorts have not become stable yet, and the difference of cumulative incidence in the current longest follow- up years is the best estimate of overdiagnosis so far. Conclusion: Overdiagnosis rates of prostate cancer in people who received screening in Finland was estimated as 2.27%,15.4%, 11.4%, and 10.2% for 1929-32, 1933-36,1937-40, and 1941-44 cohorts, respectively. / Thesis / Master of Science (MSc)

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Cancer, the second most common cause of death in the United States, is a collection of diseases caused by uncontrolled cell growth and metastasis. The main treatment for cancer is chemotherapy, which generally kills fast growing cells nonspecifically and has many side effects. A different type of cancer treatment, called targeted therapy, aims to avoid general toxicity by using drugs that block the activity of specific gene products, usually encoded by oncogenes, which have been shown to drive tumor growth. To date, targeted therapies, alone or in combination with chemotherapies, have mainly been successful in rare subsets of patients with tumors addicted to single oncogenes. This has created a rationale to mainly treat patients with an oncogene-addiction (such as those carrying mutated or overexpressed kinases) with targeted therapies like erlotinib and trastuzumab, which inhibit human epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2/ErbB2), respectively. Here, evidence is provided that targeted therapies are also effective in tumors that are dependent on multiple growth factors - a phenomenon that is called combinatorial ligand addiction. Specifically, it is shown that ligands that bind the EGFR family and the hepatocyte growth factor receptor (HGFR/MET) can activate protein kinase B (PKB/ AKT) across a broad set of cancer cell lines, suggesting that ligand signaling is redundant and widespread. It is also shown that ErbB ligands have distinct signaling dynamics and strengths, which provides a rationale for investigating each component of the ErbB signaling network. Using a systematic approach, we found that ErbB3 is an imp01tant therapeutic target even though it is not overexpressed and lacks kinase activity. Furthermore, it is shown that cell lines with and without known oncogene-addiction express autocrine ligands and have improved growth inhibition with drug combinations that include autocrine ligand-blocking antibodies. This research demonstrates that combinatorial ligand addiction creates a new rationale for therapeutic combinations to improve efficacy and prevent resistance in cancer cells that are treated with current targeted drugs. / 2999-01-01

D. Litt et Phil. / It is well known that high levels of anxiety and/or depression often accompany the diagnosis and treatment of cancer. Literature from various sources, but in particular from the fairly new field of research, Psychoneuroimmunology, also provides ample evidence that excessive anxiety and/or depression can be immunosuppressive. It makes sense, therefore, that any intervention restoring balance to the immuno-regulatory system, thereby allowing the body’s innate healing processes to focus on eliminating cancer, is highly desirable. In line with current thinking based on the mind-body connection as well as cognitive behavioural techniques utilised in many therapeutic settings, various psychological interventions have been found to help the patient gain a better sense of control over distressing symptoms and side-effects of cancer. Some of these include: basic cognitive restructuring, hypnotherapy, relaxation-meditation techniques, art and music therapy, and guided imagery. Substantial international research illustrates the beneficial effect that relaxation and/or guided imagery provides in such diverse settings including work, sport and health. In this regard, it was decided to run a pilot study to ascertain whether a customised tape recording with a relaxation and guided imagery dialogue aimed at helping patients manage and cope with negative symptoms of cancer, could significantly reduce anxiety levels in patients with cancer receiving radiotherapy. To operationalise the above, 30 men and women, aged between 20 and 80, with Stages 1, 2 or 3 breast, prostrate, gynaecological cancers, and head and neck cancers, who were about to commence radical (minimum 25 fractions) radiotherapy, were randomly selected to an experimental and a control group. A consecutive sample, pre-test post-test experimental design was applied to this study in which the experimental and control groups were subjected to pre- and post radiotherapy Hospital Anxiety & Depression (HAD) Scale, Institute for Personality Assessment and Training (IPAT) Anxiety Scale and blood pressure measurements during their 1st, 3rd, 6th week cycle of treatments, as well as a final measurement 12 weeks after commencement of therapy. The main hypothesis of this pilot study was that there would be statistically significant decreases in levels of anxiety as a result of the intervention of guided imagery tape recording in patients with non-metastatic cancer undergoing curative radiotherapy. For the intervention, each experimental participant was taught a relaxation technique and then following an interview a customised guided imagery dialogue developed for the participant’s sole use. The participant was requested to listen to this tape at least once a day. The control group had the same pre- and post tests as the experimental group, but did not receive any intervention. Statistical analysis of the data revealed that the experimental group showed a tendency towards decreased blood pressure and anxiety over the course of radiotherapy. The most significant change, however, was noted in terms of diastolic blood pressure, suggesting that the intervention corresponded to a physiological decrease in anxiety. There was not a statistically significant difference in terms of the measured psychological variables. A general conclusion to this pilot study suggests that whilst guided imagery may contribute to a lowering of anxiety, additional cognitive intervention would probably affect a more substantial and sustained change in the patient. Although this pilot study revealed some methodological weaknesses the results are sufficiently encouraging to warrant further in-depth research regarding the use of guided imagery as a cost-effective, easy method for individuals to learn and utilise as part of their integrative cancer treatment programme.

dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements of the degree of Master of Science. Johannesburg, 2014 / Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting the mRNA of LRP using siRNA-LAMR1 by 100, 44% and 73%, respectively. This knockdown of LRP expression resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay by 72%, 52% and 45% in WHCO, MCF-7 and MDA-MB 231 cells, respectively. Moreover, the reduction in cellular viability was accompanied by a significant decrease in cell proliferation by 26%, 43% and 59% in MCF-7, WHCO1 and MDA-MB 231 cells, respectively. The exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells as detected by the Annexin-V/7-AAD assay indicates that the reduction in cellular proliferation and viability is due to the induction of apoptosis which is elaborated by the loss of membrane symmetry as well as observations of nuclear morphological changes in all cell lines post transfection with siRNA-LAMR1. This data indicates that LRP/LR is crucial for the maintenance of cellular viability of cancerous cells and our findings recommend siRNA technology as a novel alternative therapeutic approach for the treatment of metastatic cancer.

Orientador: Debora Aparecida Pires de Campos Zuccari / Banca: Dorotéia Rossi Souza / Banca: Flavia Cristina Rodrigues Lisoni / Banca: Marília de Freitas Calmon / Banca: Wilson Araújo da Silva Filho / Resumo: O câncer de mama apresenta altas taxas de incidência e também de mortalidade, sendo a neoplasia mais comum entre as mulheres. MicroRNAs (miRNAs) são pequenas moléculas de RNAs não codificantes que desempenham papel fundamental na regulação gênica. Estudos recentes têm demonstrado que miRNAs estão diretamente envolvidos na iniciação e progressão de vários tipos tumorais, incluindo o câncer de mama. Diversos miRNAs têm sido descritos como promotores ou supressores tumorais, podendo estar associados ao crescimento do tumor e metástase. Atualmente, tem sido demonstrado que a administração exógena da melatonina, um hormônio naturalmente secretado pela glândula pineal, apresenta diversos efeitos oncostáticos em diferentes tipos tumorais. Assim, o objetivo do presente estudo foi avaliar o papel da melatonina em uma possível via metastática envolvendo a regulação de miRNAs em células da linhagem de câncer de mama metastática e triplo-negativa MDA-MB-231. Inicialmente, a expressão de 384 miRNAs foi avaliada utilizando placas "Taqman Low-density Array" (TLDA). Para futuras validações, foram selecionados apenas miRNAs que apresentaram fold change >1,5 e <0,5. Os resultados demonstraram que a melatonina modulou a expressão de 17 miRNAs (11 superexpressos e 6 inibidos). Dentre os miRNAs modulados pela melatonina, miR-148b e miR-210 foram confirmados por qRT-PCR, selecionados e utilizados para investigações funcionais. As células MDAMB-231 foram então transfectadas para inibição... / Abstract: Breast cancer has high rates of incidence and mortality, and it is the most common cancer among women. MicroRNAs (miRNAs) are small molecules of non-coding mRNA that play a key role in gene regulation. Recent studies have shown that miRNAs are directly involved in the initiation and progression of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors of metastasis and may be associated with tumor growth and metastasis. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. Herein, we investigated if the antimetastatic effects of melatonin were coordinated by miRNAs involved in tumor progression. The expression of 384 miRNAs was measured using Taqman Low-density Array (TLDA) cards. Considering the cut-off we imposed (fold change >1.5 and (fold change >1.5 and<0.5) were evidenced the modulation of 17 miRNAs (11 up and 6 down). Among all miRNAs modulated, the selected miR-210 and miR-148b were further confirmed by qRT-PCR and tested for functional investigations. First, we engineered cells for miR-210 or miR-148b overexpression or depletion (stable or transient), then we evaluated the effect of melatonin on c-Myc protein expression and migration. Melatonin reduced c-Myc expression and migration in cell depleted or not for miR-148b. However no effect on c-Myc or migration was observed for cells depleted for miR-148b when ... / Doutor