After numerous renewals and reignitions since the initiation of the "War on Cancer" more than five decades ago, the recent reignition of "Moonshot to Cure Cancer" points to the systemic persistence of cancer as a major cause of loss of life and livelihood. Literature points to the diminishing returns of cancer research through time, as well as heterogeneities in cancer research centers' innovation strategies. This dissertation focuses on the strategic decision by cancer research centers to invest their resources in conducting early phases of clinical trials on new candidate drugs/treatments (resembling exploration) or late phases of clinical trials that push established candidates towards acquiring FDA approvals (resembling exploitation). The extensive clinical trials data suggests that cancer research centers are not only different in their emphasis on exploratory trials, but also in how their emphasis is changing over time. This research studies the dynamics of this heterogeneity in cancer research centers' innovation strategies, how experiential learning and capability development interact to cause dynamics of divergence among learning agents, and how the heterogeneity among cancer research centers' innovation strategies is affected by the dynamics of learning from experience and capability development. The findings of this dissertation shows that endogenous heterogeneities can arise from the process of learning from experience and accumulation of capabilities. It is also shown that depending on the sensitivity of the outcome of decisions to the accumulated capabilities, such endogenous heterogeneities can be value-creating and thus, justified. Empirical analysis of cancer clinical trials data shows that cancer research centers learn from success and failure of their previous trials to adopt more/less explorative tendencies. It also demonstrates that cancer research centers with a history of preferring exploratory or FDA trials have the tendency to increase their preference and become more specialized in one specific type (endogenous specialization). These behavioral aspects of the cancer research centers' innovation strategies provide some of the tools necessary to model the behavior of the cancer research efforts from a holistic viewpoint. / Doctor of Philosophy / The "Moonshot to Cure Cancer" was renewed most recently in September 2022. However, renewal and reignition of this national collective effort is nothing new; this effort started as "War on Cancer" in 1971 and has been reignited numerous times. After more than 50 years of our collective battle to cure cancer, it claims almost 600,000 lives annually and remains as the second leading cause of death in the US. There are a wide variety of cancer research centers from all around the world contributing to this collective effort and they make considerably different decisions regarding their investment in research. There is evidence suggesting that some of the research centers' investment decisions are not optimal and can be improved. It has been shown that systems such as patent regulations can be revised to encourage such improved decisions among cancer research centers. This dissertation focuses on the process of clinical trials for new drugs/treatments for cancer. New drugs/treatments have to pass different phases of trials to ensure that they are safe and effective before they can acquire FDA approvals. Cancer research centers decide whether to invest in early phases of clinical trials for new drug/treatment candidates or invest in late phases of trials for candidates that have already passed the early phases. The clinical trials data show that there has been a sharp rise in number of early phases of trials on new drugs/treatments; however, the same rise cannot be seen in the late phases of trials resulting in approvals. It can also be seen that different research centers put different levels of emphasis on initiating early phases of trials for new drugs/treatments (exploration). In this dissertation, the hypothesis is that this ongoing dilemma that cancer research centers face to invest on how much emphasis to put on exploration in their clinical trials is affected by learning from experience. To test this hypothesis, a mathematical model is used to show differences in decisions can be causes solely by learning from experience, when the decision maker is learning "what to do" from success/failure of previous efforts and learning "how to do it" from practicing and accumulating the required skills. Then, the hypothesis is formally tested using the clinical trials data. The results show that cancer research centers learn from the success and failure of their previous exploratory trials when deciding on their emphasis on exploration. Also, they accumulate skills, resources, and capabilities relevant to the type of research the conduct more often and specialize in either of late- or early-phases of trials. The findings of this dissertation show that learning from experience can cause in differences in decisions. It also finds evidence that cancer research centers learn to place different levels of emphasis on exploration in their clinical trials. These findings can later be used in models of the cancer research ecosystem to study how funding structures and policies can be changed to improve the outcomes of our collective effort to cure cancer.

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Das entwickelte Bildgeführte Präzisionsbestrahlungsgerät für Kleintiere (engl. Small Animal Image-Guided Radiation Therapy – SAIGRT) dient der schnellen, hochauflösenden Röntgenbildgebung und präzisen, konformalen Bestrahlung von Kleintieren im Rahmen präklinischer in-vivo Experimente für die translationale Krebsforschung. Speziell programmierte Softwares zur Gerätesteuerung sowie zur Bildkorrektur- und Bildrekonstruktion auf dem zentralen leistungsfähigen Arbeitsplatz-PC stellen alle Gerätefunktionen zur Verfügung und ermöglichen durch automatisierte Abläufe und intuitive grafische Nutzeroberflächen eine einfache, sichere Bedienung. Für die Bestrahlungsplanung wird eine vollwertige, aus der humanen klinischen Strahlentherapie adaptierte 3D-Bestrahlungsplanungssoftware eingesetzt, die etablierte Werkzeuge für den Transfer und die Koregistrierung multimodaler Bilddaten, die Konturierung und Segmentierung von Zielvolumina und Risikoorganen sowie die Erstellung und Validierung von Bestrahlungsplänen enthält. Die resultierende Dosisverteilung wird darin basierend auf dem individuellen CT-Datensatz des Versuchstieres und einem auf das SAIGRT angepassten Maschinenmodell mittels eines Monte-Carlo-Algorithmus exakt und realitätsnah simuliert. Durch geometrische Kalibrierungen und vielfältige Basisdatenmessungen für die Bildgebung und Bestrahlung im Rahmen der Gerätekommissionierung ist eine Zielgenauigkeit von ca. ±0,1 mm mit hoher geometrischer Abbildungstreue und guter Bildqualität bei Bildgebungsdosen vergleichbar denen klinischer Radiografie- und CT-Geräte möglich. Die Dosisverteilung zur Bestrahlung der Versuchstiere spiegelt bei der definierten Strahlungsqualität größenskaliert die humane Strahlentherapie mit hochenergetischer Photonenstrahlung von klinischen Linearbeschleunigern wider. Ein umfassendes Qualitätssicherungsprogramm bestehend aus regelmäßiger Wartung und wiederkehrenden Konstanzprüfungen der Bildgebung und Bestrahlung sichert dauerhaft den technisch einwandfreien Zustand und die ordnungsgemäße Verfügbarkeit aller Gerätefunktionen in gleichbleibender Güte. Das SAIGRT ist somit nachweislich geeignet, bildgeführte Bestrahlungen mit einem Ablauf analog dem einer modernen klinischen Strahlentherapie am Menschen in präklinischen in-vivo Experimenten präzise an Kleintieren zu applizieren. Es leistet dadurch einen essentiellen Beitrag zur translationalen Krebsforschung in Dresden, indem die klinische Situation realistischer modelliert und so potenziell die Übertragbarkeit der Ergebnisse auf Krebspatienten verbessert werden kann. / The Small Animal Image-Guided Radiation Therapy (SAIGRT) platform facilitates fast, high resolution X-ray imaging and precise, conformal irradiation of small animals in preclinical in-vivo experiments for translational cancer research. Dedicated software for device control as well as image correction and reconstruction on a central high performance PC provide all device functions and allow simple and safe operation by automated procedures and intuitive graphical user interfaces. A fully 3D treatment planning software adapted from human clinical radiation therapy is used for treatment planning, containing established tools and methods for the transfer and registration of multimodality imaging data, contouring and segmentation of target volumes and organs at risk as well as creation and evaluation of treatment plans. Based on an individual CT scan of the small animal and a machine model adapted for the SAIGRT, the resulting dose distribution is simulated by a Monte-Carlo algorithm in a precise and realistic manner. Geometrical calibrations as well as manifold basic data measurements for X-ray imaging and irradiation during commissioning resulted in a targeting and imaging accuracy of about ±0.1 mm, a correct representation of imaging geometry and a good image quality with imaging doses comparable with those of clinical radiography and CT systems. Dose distribution of the defined beam quality used for irradiation of small animals reflects a downsized human radiation therapy using high energy photon beams of clinical linear accelerators. A comprehensive quality assurance program comprising regular maintenance and periodic constancy tests of X-ray imaging and irradiation ensures permanent technically perfect condition and proper availability of all implemented functions in a stable high quality. The SAIGRT platform is feasible for image-guided irradiations precisely applied to small animals in preclinical in-vivo experiments using a workflow of modern human radiation oncology. Thus, it significantly contributes to translational cancer research by more realistic modelling the clinical situation and potentially brings the results closer to their clinical implementation.

In the medical field, the applications of ultraviolet (UV) radiation are limited to skin or reachable sites due to its low penetration depth into biological tissue. Contrary to UV radiation, X-rays can penetrate the body with almost no attenuation, but they result in toxic side effects. Inorganic scintillators absorb X-rays and convert them into UV or visible photons and are usually used for medical imaging. We propose the use of high density inorganic nanoscale scintillators with the ability to absorb externally applied ionizing radiation directly at the site of application, e.g., inside a tumor, and to convert this ionizing radiation into UV photons in situ, enabling new biomedical applications inside the body. In this thesis, two specific new biomedical applications are discussed in detail: The first application is the use of UV-B emitting nanoscale scintillators for highly localized drugs released or activation of photoactivable therapeutics using only X-rays. The second novel approach is the use of UV-C emitting nanoscale scintillators as radiation sensitizers. However, size-reduction of inorganic scintillators, and most inorganic phosphors in general, usually result in quenching of the photoluminescence properties, defects on the surface of the particles, and a decrease of radiation hardness. Colloidal solutions of nearly monodisperse LaPO4:Gd nanocrystals (5nm) were shown to strongly emit UV radiation upon excitation with X-rays or vacuum UV radiation (160nm). The UV emission of the particles consisted mainly of a single line at 311nm. This UV-B emission of the particles was used to excite the fluorescence of laser dyes dissolved in the colloids. The emission of the dyes was also observed in the case of high dye concentrations, proving that the concept of using radiation with a high penetration depth (X-rays) to excite fluorescence emission with a low penetration depth (UV-B) wavelength is feasible. Pr-doped LuPO4 emits UV-C radiation between 225 and 280nm, where DNA shows strong absorption bands. Therefore, a systematic study of the luminescence of LuPO4:Pr was performed: Different doping concentrations, particle sizes, and excitation sources were compared. Furthermore, it was found that Pr and Nd co-doped LuPO4 results in increased UV-C emission independent of excitation source due to energy transfer. The highest UV-C emission intensity was observed for LuPO4:Pr,Nd(1%,2.5%) upon X-ray irradiation. Finally, LuPO4:Pr,Nd nanoparticles were synthesized, and the biological efficacy of the combined approach (X-rays and UV-C) was assessed using the colony formation assay. Cell culture experiments confirm increased cell death compared to X-rays alone due to the formation of UV-specific DNA damages, supporting the application of the herein synthesized particles as radiation sensitizers.

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Indiana University-Purdue University Indianapolis (IUPUI) / Resveratrol has been shown to inhibit cell growth and induce apoptosis, as well as augment chemotherapeutics and irradiation in multiple cancer types. However, it is unknown if resveratrol is beneficial for treating drug-resistant cancer cells. To study the effects of resveratrol in triple negative breast cancer cells that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the MDA-MB-231 breast cancer cell line. The resulting cell line, MDA-MB-231/PacR, exhibited a 12-fold increased resistance to paclitaxel but remained sensitive to resveratrol treatment. Resveratrol treatment reduced cell proliferation and colony formation and increased senescence and apoptosis in both the parental MDA-MB-231 and MDA-MB-231/PacR cell lines. Importantly, resveratrol treatment augments the effects of paclitaxel in both cell lines. The expression of the drug efflux transporter gene, MDR1, and the main metabolizing enzyme of paclitaxel gene, CYP2C8, was increased in the resistant cells. Moreover, pharmacological inhibition of the protein products of these genes, P-glycoprotein and CYP2C8, decreased paclitaxel resistance in the resistant but not in the parental cells, which suggests that the increase of these proteins are important contributors to the resistance of these cells. In conclusion, these studies imply that resveratrol, both alone and in combination with paclitaxel, may be useful in the treatment of paclitaxel-sensitive and paclitaxel-resistant triple negative breast cancers.

Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Cancer stem cells (CSCs) are proposed to play a major role in tumor progression, metastasis, and recurrence. The Human Epidermal growth factor Receptor 2 (HER2) gene is amplified and/or its protein product overexpressed in approximately 20% of breast cancers. HER2 overexpression is associated with increased CSCs, which may explain the aggressive phenotype and increased likelihood of recurrence for HER2+ breast cancers. Telomerase is reactivated in tumor cells, including CSCs, but has limited activity in normal tissues, providing support for the use of telomerase inhibition in anti-cancer therapy. Telomerase inhibition via an antagonistic oligonucleotide, imetelstat (GRN163L), has been shown to be effective in limiting cell growth in vitro and limiting tumor growth. Moreover, we have previously shown imetelstat can decrease metastases to the lungs, leading us to question if this is due to imetelstat targeting the CSC population. In this thesis, we investigated the effects of imetelstat on CSC and non-CSC populations of HER2+ breast cancer cell lines, as well as a triple negative breast cancer cell line, which lacks HER2 overexpression. Imetelstat inhibited telomerase activity in both CSC and non-CSC subpopulations. Moreover, imetelstat treatment alone and in combination with trastuzumab significantly reduced the CSC fraction and inhibited CSC functional ability, as shown by a significant decrease in mammosphere counts and invasive potential. Tumor growth rate was slower in combination treated mice compared to either drug alone. Additionally, there was a trend toward decreased CSC marker expression in imetelstat treated xenograft cells compared to vehicle control. The decrease in CSC marker expression we observed occurred prior to and after telomere shortening, suggesting imetelstat acts on the CSC subpopulation in telomere length dependent and independent mechanisms. Our study suggests addition of imetelstat to trastuzumab may enhance the effects of HER2 inhibition therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.