<p> <i>Sushi Domain Containing 2 (SUSD2)</i> encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. A clinically annotated HGSOC tissue microarray was stained with an anti-SUSD2 antibody. Patients with tumors that had weak SUSD2 staining had a shorter median survival (31.7 months) compared to patients that had tumors with strong SUSD2 staining (49.1 months; p value = 0.0083).</p><p> To investigate the role of SUSD2 in HGSOC, stable OVCAR3, OVSAHO and KURAMOCHI cell lines were established through transfection of shRNA targeted to <i>SUSD2</i> transcripts (SUSD2 knock-down [KD] cell lines) or non-targeting shRNA (SUSD2 NT) cell lines. Boyden chamber and wound healing assays demonstrated that OVCAR3, OVSAHO and KURAMOCHI SUSD2 KD cells migrated at significantly higher rates than their SUSD2 NT counterpart cell lines. RT-qPCR and western immunoblot analysis indicated an inverse relationship between SUSD2 and well characterized mesenchymal genes, such as <i>TWIST1, ZEB1</i> and <i>CHD2.</i> In addition, OVCAR3 and KURAMOCHI SUSD2 KD spheroids displayed increased mesothelial clearance ability compared to SUSD2 NT spheroids.</p><p> To explore the potential for SUSD2 to inhibit late-stage HGSOC metastasis, female athymic nude mice were injected with either OVCAR3 NT or OVCAR3 KD cells. Fewer nodules were observed in the pancreas and omentum of the OVCAR3 NT mice when compared to the OVCAR3 KD mice. Furthermore, OVCAR3 KD mice had a significantly shorter median survival compared to OVCAR3 NT mice (175 days compared to 185.5 days, respectively; p-value = 0.0047).</p><p> KURAMOCHI lysate was immunoprecipitated for SUSD2-associated immunocomplexes and subjected to liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis, yielding a list of candidate SUSD2-interacting proteins associated with RNA processing. Immunofluorescence analysis of OVCAR3, KURAMOCHI and SKBR3 cells and western immunoblot analysis of their subcellular extracts revealed SUSD2 to be present in cell nuclei, mitochondria and cytoplasm; however, SUSD2 was relatively less abundant in SKBR3 nuclei.</p><p> Our findings suggest that increased <i>SUSD2</i> expression in HGSOC impedes metastasis, consistent with prolonged survival observed in HGSOC patients with high <i>SUSD2</i>-expressing primary tumors. The differences in subcellular distribution between HGSOC cells and breast cancer cells may explain alternate functions of SUSD2 in different cancers.</p><p>

<p> Durotaxis is a mechanism of directed cell migration in which cells respond to gradients of extracellular stiffness. While durotaxis has been predominantly characterized in a subset of mesenchymal cells, the potential for cancer cells to durotax has not been well defined. Numerous studies have demonstrated the role of diffusible factors in cancer cell migration and metastasis. However, given the gradual stiffening of many tumor microenvironments, we hypothesized that a durotactic mechanism might also contribute to the migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing a stiffness gradient that mirrors the physiological stiffness encountered by cells throughout a variety of tissues. Customized MATLAB software permitted rapid acquisition of positional data for migrating cells. The automation of cell tracking allowed for large sample sizes and therefore a more robust statistical analysis than previously used to evaluate durotaxis. Durotaxis assays identified two glioblastoma lines, a metastatic breast cancer line, and fibrosarcoma line that are responsive to changes in extracellular stiffness. Of interest was our finding that cancer cells showed strong durotactic behavior when occupying the softest region of the stiffness gradient with decreasing responsiveness as cells occupied increasingly stiff regions of the gradient. These observations suggest that durotaxis is influenced by the stiffness of a cell&rsquo;s local environment, with soft substrates increasing durotaxis efficiency in the cancer cell lines evaluated. Furthermore, we determined that PI3K inhibition was sufficient to inhibit glioblastoma chemotaxis but not durotaxis, suggesting that alternative signaling is used to respond to durotactic directional cues. Lastly, we evaluated the protrusion and retraction dynamics of cells on a stiffness gradient and have identified a unique mode of discontinuous migration exhibited by these cells. Based on our observations, we developed a durotaxis model to suggest how discontinuously migrating cells can respond to a gradient by moving towards regions of increasing stiffness.</p><p>

<p> Radiation oncology has evolved as an advanced tool in treating chronic diseases from a mere experimental application of X-rays. These advances were made possible due to combined efforts of physicians, clinicians and information technology professionals. The Radiation Oncologist depends on clinical and information technology disciplines to solve complex health conditions. This creates demand for consulting work for information technology professionals who can design, customize and deploy software applications that are used in radiation oncology departments at hospitals. Hri-Tech Consultants, LLC a consulting firm, intends to offer consulting services to medium sized hospitals in Orange County area in design, development and deployment of radiation oncology software applications. The firm&rsquo;s unique strength in Radiation Oncology workflow coupled with Lean Six Sigma and ITIL processes will be rarely matched by other providers in the target market.</p><p>

Lysosomes, the recycling units of the cell, participate in the signaling pathway to apoptosis, which has stimulated the search for anti-cancer drugs targeting the lysosomal compartment. Lysosomes are, however, often altered in cancer cells. The aim of this thesis was to investigate the involvement of lysosomes during apoptosis in normal and cancer cells. We developed and used flow cytometric methods to measure cytosolic and lysosomal pH in cells. The cytosolic pH of U937 cells decreased, in a caspase-independent way, by 1.4 pH-units during apoptosis. Concomitantly, the lysosomal pH increased from 4.3 to 5.2, suggesting that proton release from lysosomes might be responsible for cytosolic acidification. When studying the lysosomal pH of head and neck squamous cell carcinoma (HNSCC) cell lines and normal oral keratinocytes (NOKs), the pH was significantly increased in three of five HNSCC cell lines, as compared to NOKs. Moreover, high lysosomal pH correlated to low expression of the B subunit of the vacuolar V0/V1-ATPase, a necessary component of the proton pump responsible for lysosomal acidification, and to reduced intrinsic cisplatin sensitivity. Cisplatin-induced apoptosis was, at least partly, dependent on lysosomal cathepsins. When investigating the colony formation ability of the two HNSCC cell lines LK0412 and SqCC/Y1, both were found to give rise to holoclones, indicating the presence of cells with cancer stem cell properties. Holoclone cells from the LK0412 cell line were less sensitive to cisplatin compared to more differentiated paraclone cells. Moreover, we detected differences in intracellular localization of the lysosomal compartment and expression of cathepsins between holo- and paraclone cells. This thesis shows that changes found in the lysosomal compartment of cancer cells, such as alteration of lysosomal pH, might influence the outcome of a drug treatment. In addition, differences in drug sensitivity between subpopulations of tumor cells may affect the outcome of an anticancer therapy. / Programmerad celldöd eller apoptos är en viktig mekanism för att upprätthålla balans mellan kroppens celler. Vid exempelvis cancer fungerar inte styrningen av denna process, vilket leder till att för få celler dör och en tumör kan växa ohämmat. Denna avhandling fokuserar på lysosomen, en mycket sur organell i cellen som är ansvarig för nedbrytning av cellmaterial. Hos cancerceller är lysosomerna ofta förändrade. Vi har undersökt lysosomernas roll under apoptos hos normala celler och hos cancerceller. För att kunna undersöka pH-förändringar under apoptos har vi utvecklat metoder att mäta cytosoliskt och lysosomalt pH med hjälp av en teknik som kallas flödescytometri. I apoptotiska celler ser vi att det cytosoliska pH:t sjunker med 1.4 pH-enheter till pH 5.7 samtidigt som det lysosomala pH:t ökar från 4.3 till 5.5. Detta tyder på att läckage av vätejoner från lysosomerna kan orsaka en försurning av cytosolen under apoptos. Genom att studera normala orala keratinocyter och jämföra dessa mot fem olika cellinjer eeablerade från skivepitelcancer från munhåla har vi också funnit ett samband mellan det lysosomala pH:t och känsligheten för cellgiftet cisplatin. Cisplatinbehandling leder till apoptos hos alla celler men en högre dos krävs hos celler som har ett högt lysosomalt pH. Tumörer tros innehålla ett litet antal sk cancerstamceller, som har förmåga att kontinuerligt kopiera sig själva utan att åldras. Överlevnad av dessa celler tros vara orsaken till att en tumör återkommer efter en behandling. Vi visar i denna avhandling att cellinjer från skivepitelcancer innehåller celler som har cancerstamcellsegenskaper, och att dessa celler kan ha en lägre känslighet mot cisplatin jämfört med mer utvecklade cancerceller. Lysosomerna utgör ett intressant framtida mål för nya cancerläkemedel. I denna avhandling visar vi att förändringar i det lysosomala systemet kan påverka effekten av ett läkemedel och att skillnader mellan olika sub-populationer av celler från samma tumör kan påverka resultatet av en behandling.

<p> Pancreatic cancer is the one of the leading causes of cancer-related deaths in the United States and is characterized with low survival rate and high drug resistance. Because of the redundant and highly mutated signaling pathways in pancreatic cancer, numerous combinational therapies have been sought. Currently the selection of drug combinations is largely empirical and methods of evaluating and optimizing drug combinations have not been standardized. An important reason for this is the lack of comprehensive characterization of drug mechanisms of action and causes for drug resistance. </p><p> The purposes of this dissertation are: first, to set up a paradigm for evaluating drug combinations mathematically and translating the evaluation methods from <i>in vitro</i> to <i>in vivo</i> preclinical systems; second, to serve as an example for characterizing the biological signaling pathways and drug pharmacology comprehensively with systems modeling approaches, supported with &ldquo;big data&rdquo; from advanced techniques such as proteomic analysis; and third, using such systems models, further selecting and optimizing drug combinations to reverse drug resistance and enhance efficacy. </p><p> The two drugs selected are gemcitabine, a major component in the therapies for pancreatic cancer treatment, and birinapant, an antagonist of inhibitor of apoptosis proteins (IAP). In Chapter 1, the efficacy of this drug combination was evaluated in PANC-1 cells. A basic pharmacodynamic (PD) model was developed to characterize the temporal changes in the numbers of attached and floating cells after treatments, and synergistic effects were observed for both proliferation inhibition and death induction. Measurements of cell cycle distributions and apoptosis were then obtained and a mechanism-based PD model was developed to reveal more details and capture the major features of the beneficial interactions. From the mechanism-based PD model, different exposure schedules were tested and an optimal one to achieve maximal efficacy was proposed. </p><p> Assumptions were made in developing the mechanism-based PD model in Chapter 1. In Chapter 2, a proteomic approach was utilized for a comprehensive, unbiased study of proteins perturbed by gemcitabine and birinapant to test previous hypotheses. The mechanisms of action for both drugs were characterized more intensively, and additional details were incorporated into the interaction knowledge described previously. Based on the proteomics data, reasons for gemcitabine resistance were discussed, and regulators of DNA damage responses involving DNA repair, anti-apoptosis, and pro-migration and invasion proteins were proposed as promising candidates for therapeutic targeting. </p><p> With the rich quantitative proteomics data, a network modeling approach was attempted in Chapter 3. Quantitative relationships were developed for selected signaling pathways of cell cycle regulation, DNA damage responses, DNA repair, apoptosis, NF-&kappa;B, and MAPK-p38, which were then linked to describe the cell cycle progression and apoptosis, and finally to changes in cell numbers. Based on the developed network model, simulations were made under different conditions and compared with observations, serving as a validation process. The impact of p53 mutation and p53 silencing on the efficacy of gemcitabine was tested with this model. Sobol Sensitivity Analysis was applied to select promising targets to be combined with gemcitabine. In addition, the efficacy of curcumin combined with gemcitabine was evaluated based on the model simulation. </p><p> With extensive evaluation and comprehensive characterization of the mechanisms of this drug combination in cell culture, efforts were continued to investigate the effects of the combination in a mice xenograft model. In Chapter 4, pharmacokinetic information for gemcitabine and birinapant was gathered from the literature and full physiologically-based pharmacokinetic models (PBPK) were developed to characterize drug distribution in the body and into the pancreatic tumor. The tumor concentrations then were used to drive inhibition in tumor growth and a semi-mechanistic PBPK/PD model was developed to evaluate the efficacy of the drug combination <i>in vivo.</i> Their joint effects were revealed as merely additive. The network model developed in Chapter 3 was introduced to bridge the PBPK and PD models, and reasons for the discrepancies <i> in vitro</i> and <i>in vivo</i> were explored. Model predictions showed that simultaneous dosing was preferable to sequential dosing <i> in vivo</i> with stronger suppression of the DNA repair signaling. </p><p> In summary, this dissertation proposed a paradigm for evaluating drug combinations quantitatively in preclinical systems of cell lines and xenograft models. Comprehensive characterization of drug mechanisms of action and biological systems through network modeling can facilitate the selection and optimization of candidates for anti-cancer combination therapy. The bridging of knowledge in different scales with mathematical models in different complexity helps to minimize the gap of translating from <i>in vitro</i> to <i> in vivo</i> or even from preclinical to clinical research.</p><p>

<p> There are approximately 230,000 new diagnoses of prostate cancer every year in the U.S., making prostate cancer the most diagnosed cancer in men. It is responsible for approximately 30,000 deaths per year, with only lung cancer taking more lives. An important distinction must be made in men with prostate cancer. The majority of men with prostate cancer have a relatively indolent form of the disease, meaning high survival rates (100% survival 5 years after diagnosis) and no invasion of the tumor to other organs. However, approximately 4% of men are diagnosed with an aggressive form of the disease, and for these men, the survival rate is a mere 30% after 5 years. And for many patients, it is clinically difficult to differentiate between the indolent and the aggressive forms of the disease. Therefore, it is imperative to develop new genetic models of prostate cancer, and the mouse is an excellent model organism in which to do so. In 2009, mice were used to discover a new type of stem cell, called a castration-resistant Nkx3.1-?expressing cell in the luminal cell population of the prostate. We have used a mouse model targeting these cells to study the roles of two tumor suppressors, adenomatous polyposis coli (Apc) and Smad4. Apc down-regulates the Wnt signaling pathway, which is a carcinogenic pathway in the prostates of humans and mice. Deletion of Apc in mice causes an increase of Wnt signaling and prostate cells to proliferate but not invade, which represents a relatively indolent, precancerous phenotype. Smad4 is a transcription factor that controls the signaling of two pathways: transforming growth factor &beta; and bone morphogenetic protein signaling. Deletion of Smad4 causes these pathways to shut off. When Apc and Smad4 are deleted simultaneously, mice develop aggressive, invasive prostate cancer. This work suggests that these two tumor suppressors &ndash; and the pathways they control &ndash; are important regulators of prostate cancer, could allow for clinicians to differentiate between indolent and aggressive disease, and should be targeted therapeutically in prostate cancer patients.</p><p>

<p> Cancer encompasses a broad range of complex malignancies characterized by diverse sites of origin, genetic landscapes, and disease progressions. This diversity dictates the manner in which these cancer cases are clinically managed, as well as the overall prognosis. Despite these differences, most cancers exhibit universal hallmarks that contribute to tumor growth and cell survival. As such, these hallmarks are the focus of many research efforts and represent high priority targets for anticancer therapy. While some novel treatment regimens targeting these features have provided good outcomes, effective therapeutics for aggressive cancers are still needed. </p><p> To that end, I used patient- derived xenografts (PDX) of colorectal cancer (CRC) and an established cell line of advanced prostate cancer (CaP) to target oncogenic drivers, specifically mTOR and MEK, and altered metabolism for decreased tumor cell growth. My work defined genomic landscapes in CRC for which molecularly targeted agents (against MEK alone and in combination with mTOR) were most effective. My research also revealed the glycolytic nature of CaP which, when inhibited, upregulated autophagy. Combined inhibition of glycolysis and autophagy reduced CaP cell viability, thereby supporting autophagy&rsquo;s role in cell survival during times of metabolic stress. </p><p> Chapter 1 provides an introduction on the hallmarks and history of cancer to frame the work described in Chapters 2 and 3. Chapter 2 presents the development of clinically relevant PDX models of CRC, and their sensitivity to targeted therapies. Chapter 3 describes the metabolic characteristics of CaP and the effects of inhibiting glycolysis. Chapter 4 contains unpublished data that interrogates the role of autophagy in CaP. Finally, chapter 5 presents conclusions from this work and future research directions.</p><p>

<p> The word "cancer" rarely stands alone, usually prefaced with its anatomical location: lung cancer, prostate cancer, brain cancer. With the advancement of high-throughput omics approaches, specific oncogenic events are reorganizing the landscape of cancer classification, at once creating commonalities between cancers arising in diverse anatomical locations and dividing organ-centric classifications of cancer into a multitude of subtypes. The term "precision medicine" postulates that these new, data-driven groupings based on molecular characterization are the key to making rational therapeutic choices. </p><p> The majority of this dissertation addresses the disconnect between extensive molecular characterization and poor cancer therapy outcomes for patients with glioblastoma multiforme (GBM). Despite clear evidence that hyperproduction of the ligand for PDGFR (platelet-derived growth factor receptor &alpha;) is sufficient to generate GBM of the proneural subtype, anti-PDGFR&alpha; therapeutics have proven disappointing in clinical trials. Cell adaptation contributes to therapeutic escape. In GBM, proneural tumor cells adopt transcriptional profiles of the mesenchymal subtype. The interconversion between the proneural and mesenchymal transcriptional classes within a tumor population presents both a challenge and an opportunity for therapeutic approaches. The proneural subtype has a proliferation phenotype and presents druggable targets such as PDGFR&alpha;. The mesenchymal subtype presents an invasive phenotype, but the targets are more challenging to drug. The typical screening for combination therapies that synergize to induce cell death is not as advantageous here, where the disease management is expected to include cytostatic drugs that act on two different aspects of the phenotype: proneurally mediated proliferation and mesenchymally mediated invasion. This work examines the applicability of a combination approach against a proneural target, PDGFR&alpha;, and mesenchymal targets in the STAT3 (signal transducer and activator of transcription 3) pathway, in the context of a proneural model of GBM.</p><p> The work is concluded with collection of work applying precision medicine in other disease contexts, most notably canine lymphoma.</p><p>

<p> Improvements in anticancer pharmacotherapy over the past 40 years have led to a steady increase in the number of cancer survivors worldwide. The clinical effectiveness of anticancer agents like the microtubule-stabilizing agent, paclitaxel, ultimately led to their adoption into standard of care regimens for most cancers. What makes these drugs so effective is how they bind to their respective targets to disrupt fundamental cellular processes. For example, by binding to &beta;-tubulin, paclitaxel induces polymerization and stabilization of cellular microtubules, leading to impairments in cellular functions like mitosis and intracellular transport. While an ingenious approach to kill cancer cells, microtubules are ubiquitous in all cell types. Consequently, paclitaxel has a burdensome side effect profile due to its effects on noncancerous cells. The most prevalent nonhematologic side effect is chemotherapy-induced peripheral neuropathy, which arises due to paclitaxel-induced damage to peripheral afferent sensory neurons. Cancer patients with peripheral neuropathy often develop debilitating neuropathic pain and numbness that diminish everyday quality of life. Symptoms intensify as treatment progresses and can persist for months and years beyond treatment. There is no effective treatment or prevention for chemotherapy-induced peripheral neuropathy. Instead, patients must dose de-escalate or discontinue life-saving chemotherapy, which subsequently worsens cancer prognosis. Thus, there is a compelling need to identify novel therapeutic options to prevent or treat chemotherapy-induced peripheral neuropathy so to improve both anticancer treatment and patient quality of life. The goal of this dissertation is to evaluate the neuroprotective efficacy of a first-in-class stimulator of nicotinamide phosphoribosyltransferase, P7C3-A20, against paclitaxel-induced peripheral neurotoxicity. Nicotinamide phosphoribosyltransferase is the rate-limiting enzyme in the salvage pathway for nicotinamide adenine dinucleotide, an indispensable redox biomolecule that drives energy production. We first developed an aggressive model of paclitaxel-induced peripheral neuropathy in adult male rats. Treatment with a near maximally-tolerated dose of paclitaxel produced significant, but recoverable weight loss and leukopenia. Paclitaxel-treated rats exhibited differentially altered nociceptive thresholds to noxious stimuli, including the development of persistent allodynia to mechanical and cold stimulation as well as transient hyposensitivity to heat stimulation. Toxicity associated with paclitaxel treatment required that 25% of the rats be removed from the study. Histological analysis determined that paclitaxel triggered degeneration of intraepidermal nerve fibers and up-regulated expression of activating transcription factor 3 in nuclei of neuron cell bodies residing in the lumbar dorsal root ganglia. Remarkably, daily treatment with P7C3-A20 prevented mechanical allodynia and heat hypoalgesia, and reduced the cold allodynia associated with paclitaxel treatment. P7C3-A20 also prevented intraepidermal nerve fiber degeneration and partially decreased activating transcription factor 3 expression in lumbar dorsal root ganglia neurons. A randomized, double-blind trial determined that P7C3-A20, and another analogue, P7C3-S321, dose-dependently decreased paclitaxel-induced neuropathic pain and intraepidermal nerve fiber loss. Furthermore, P7C3-A20 improved indices of general health and prevented premature death that normally arose because of overt toxicity caused by paclitaxel. P7C3-A20 displayed superior neuroprotective efficacy, while an inhibitor of poly(adenosine diphosphate-ribose) polymerase was completely ineffective. P7C3-A20 stimulated nicotinamide adenine dinucleotide production in vitro following induction of damage with either hydrogen peroxide or paclitaxel, but not under normal conditions. Additionally, P7C3-A20 in vivo stimulated nicotinamide adenine dinucleotide recovery in the hindpaw and sciatic nerve of rats treated with paclitaxel. FK866 blocked P7C3-A20-mediated nicotinamide adenine dinucleotide production in vitro and the neuroprotective effects on peripheral nociceptive neurons in vivo. Although treatment with either nicotinamide or a subthreshold dose of P7C3-A20 alone was ineffective, the combination produced neuroprotection against paclitaxel that was equivalent to that of a maximal dose of P7C3-A20. We also investigated the effects of P7C3-A20 on cancer cell proliferation and on the anticancer efficacy of paclitaxel. Only MDA-MB-231 breast cancer cells demonstrated a slight increase in proliferation by P7C3-A20, but enhanced growth of implanted MDA-MB-231 tumor xenografts was not observed. Furthermore, P7C3-A20 did not diminish the antiproliferative effects of paclitaxel, despite preventing the development of mechanical allodynia in the tumored mice. In conclusion, these studies discovered robust neuroprotective efficacy of P7C3-A20 against paclitaxel -induced peripheral neurotoxicity, likely through the enhancement of nicotinamide phosphoribosyltransferase-mediated recovery of nicotinamide adenine dinucleotide. Based on these results, clinical investigation of P7C3-A20 as a potential treatment option for chemotherapy-induced peripheral neuropathy may be warranted. </p><p>

<p>New treatments for neuroblastoma are desperately needed; high-risk neuroblastoma patients have a less than 50% five-year survival rate despite intensive treatment. The greatest impact on improving survival rates for cancer patients in recent years is the result of a number of immunotherapeutic approaches. A proportion of high-risk neuroblastoma patients undergo spontaneous regression, possibly mediated by the immune system, indicating the potential of immunotherapies targeting neuroblastoma-associated antigens. Recombinant attenuated Salmonella vaccines (RASV) are cost-effective and have shown efficacy against a number of pathogen-associated antigens and are easily adapted for use as cancer immunotherapies. Here we cloned survivin, a neuroblastoma tumor-associated antigen into RASV expression plasmids to develop 24 RASV candidate vaccines with an array of select phenotypes. While conventional recombinant attenuated Salmonella vaccines are permanently attenuated, the RASV used here are engineered with inducible in vivo attenuation and other delayed phenotypes shown to improve immune responses. Survivin expression did not impact the growth or stability of any of the RASV constructs. Six of the constructs were tested in vivo, the RASV survived in the gut lumen, and all RASV-immunized mice produced anti-Salmonella responses. Protein/adjuvant immunized mice produced humoral and cellular survivin specific immune responses; however two independent in vivo experiments showed that no survivin specific immune responses were induced in survivin-expressing RASV immunized mice. Based on the results, a number of improvements to the future development of the vaccine are suggested.