published_or_final_version / Anatomy / Master / Master of Research in Medicine

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

The aim of this thesis is to develop a better understanding of the mechanisms and modalities exploited by breast cancer cells during invasion, paying particular attention to those mechanisms employed during cell migration in 3D spheroid culture, a context that more accurately recapitulates the complexities seen in vivo. Using a hydrogel-based model system, we investigated the roles of cellular blebs in cancer migration, developed a novel protocol that enhances optical microscopy images and facilitates high content imaging techniques including mass spectrometry imaging, and then used this new technology to investigate gain of function activity of mutant p53 in breast cancer cell migration in terms of both actomyosin contractility and in lipid abundances and distributions. Chapter 1 begins with an overview of breast cancer, an introduction to cancerous behavior and migration, a discussion of the importance of 3D cell culture and interactions of breast cancer cell and the extracellular matrix, and an overview of the various imaging techniques used in this work. In Chapter 2 we describe a novel mode of breast cancer migration that we term bleb – driven migration. This migratory mode is characterized by invasive cells that are both round and bleb bearing. This migratory mode is highly dependent on actomyosin contractility, a mechanism that is imperative to bleb formation. We show that blebs can actively attach to and rearrange the extracellular matrix via accumulations of β1 integrins at the bleb necks. We show both polarization of blebs as well as collagen alignment in regions of the cell enriched in blebs, both of which are dependent on the expression of β1 integrins by the cell. The discovery of this new migratory mode is important, as many cancers are able to overcome cancer therapies by using escape mechanisms, such as alternate migratory mechanisms. As such, developing a fuller understanding of the migratory modes used by cancer cells is vital in our fight to prevent cancer deaths. Chapter 3 tackles a significant problem associated with working in 3D spheroid culture: obtaining high quality, high resolution images. 3D samples tend to be highly refractive and poorly diffusive, and image acquisition can be severely hindered due to these factors. Further, the depth at which 3D samples can be imaged is limited by the low working distances of high numerical aperture objectives. As such, we developed a protocol that enables the sectioning of invasive spheroids, which we term DISC-3D (dual hydrogel invasive cryosectioning of spheroids in 3D). This protocol enables us to capture images that are higher in resolution and signal to background noise, removes imaging depth related constraints, and enables images to be acquired using dyes and techniques that have not previously been demonstrated in invasive 3D in vitro samples. Using this technology, Chapter 4 then examines the gain of function activity mutant p53 imparts on invasive breast cancer cells. We show that mutant p53 plays a role in increasing actomyosin contractility by promoting targeting of RhoA to the cell membrane. We also show an alteration in lipid expression in mutant p53 bearing cells, a feat that is made possible through use of the DISC-3D technology. Collectively, this work provides insights in to the invasive mechanisms exploited by breast cancer cells. We repeatedly demonstrate the importance of in vitro, 3D cell culture in the study of breast cancer migration. Using such cell culture techniques, we outline previously unknown aspects of breast cancer cellular migration both in regards to the importance of blebs and to the gain of function activity of mutant p53, the latter of which was made possible through use of the DISC-3D protocol. We argue that continued study in this area will provide insights into how breast cancer cells migrate, providing paths and new treatment strategies for preventing such migration.

In sea turtles, the study of the etiology and development of fibropapillomatosis is not fully understood. Sea turtle fibropapillomatosis is a disease characterized by the proliferation of skin fibropapillomas and occasional internal fibromas. In this study, sea turtle fibropapilloma tumor and healthy tissue samples were used to look at VEGF, BCL-2 and Bax expression. Cancer tumors have a well established pattern of protein expression that involves overexpression of vascular endothelial growth factor (VEGF), responsible for the growth of new blood vessels, and a high BCL-2 to Bax ratio that leads to uncontrolled cell proliferation. Real time PCR was used to analyze VEGF expression, and Western blot techniques were used to measure BCL-2 and Bax expression. The results indicated that expression of VEGF was not significantly higher in tumor vs. skin tissue. For the differential expression of BCL-2 and Bax, the results were not in agreement with the established levels found in cancer studies, showing no significant change in BCL-2 expression and significantly higher levels of Bax in tumor vs. healthy tissue. / by Angela Bancalari-Schmidlapp. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

The purpose of this work was to develop novel optical imaging technology and algorithms as a nondestructive method for detection and diagnosis of cancer in breast specimens. There are many ways in which the diagnosis of disease can benefit from fast and intelligent optical imaging technology. Our existing ability to provide this diagnosis depends on time-consuming pathology analysis. Optical coherence tomography (OCT) is a non-invasive optical imaging modality that provides depth-resolved, high-resolution images of tissue microstructure in real-time. OCT could provide a rapid evaluation of specimens while patients are still in the office, and has strong potential to improve the efficiency in evaluation of breast pathology specimens (biopsy or surgical). In this work, we demonstrate an imaging system to address this unmet clinical need, artificial intelligence algorithms to interpret the images, and early work towards miniaturizing the technology. We present an OCT system that achieves a line scan rate of 250kHz, meaning we can image a pathology cassette in 41 seconds, which is more than double the fastest scan rate in the field. By utilizing a multiplexed superluminescent diode (SLD) light source, which has strong noise performance over imaging speed, we achieve high resolution imaging under 5 um in tissue (axially and laterally). The system features a 1.1 mm 6-dB sensitivity fall-off range when imaging at 250 kHz. The scanner features large-area scanning with the implementation of a 2-axis motorized stage, enabling visualization of areas up to 10 cm x 10 cm (prior work visualizes 3 mm x 3mm). We showcase the results of demonstrating the performance of this system on a 100-patient clinical imaging study of breast biopsies, as well as imaging of clinical pathology specimens from the breast, prostate, lung, and pancreas in an IRB-approved study. Further, we show our work towards developing artificial intelligence (AI) for cancer detection within OCT images. Using retrospective data, we developed a type of AI algorithm known as a convolutional neural network (CNN) to classify OCT images of breast tissue from 49 patients. The binary cancer classification achieved 94% accuracy, 96% sensitivity, and 92% specificity. This framework had higher accuracy than the 88% accuracy of 7 clinician readers combined in our lab’s earlier multi-reader study. Lastly, we demonstrate a supercontinuum light source based on a 1 mm2 Si3N4 photonic chip for OCT imaging that has better performance than the state-of-the-art laser. Existing broadband laser sources for OCT are large, bulky, and have high excess noise. Our Si3N4 chip fundamentally eliminates the excess noise common to lasers and achieves 105 dB sensitivity and 1.81 mm 6-dB sensitivity roll-off with only 300 µW power on the sample.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatigue is one of the most common and debilitating symptoms reported by cancer patients, yet it is infrequently diagnosed or treated. Relatively little is understood about its etiology in the cancer context. Recently, as researchers have begun to focus attention on cancer-related fatigue (CRF), depression has emerged as its strongest correlate. Few longitudinal studies have been done, however, to determine whether causal influences between the two symptoms exist. The aim of the current study was to determine whether depression has a causal influence on CRF and whether reciprocal effects exist. The study used a single-group cohort design of longitudinal data from a randomized controlled trial (N = 405) of an intervention for pain and depression in a heterogeneous sample of cancer patients. To be eligible, participants met criteria for clinically significant pain or depression. A hypothesis that depression would influence change in fatigue after 3 months was tested using latent variable cross-lagged panel analysis, a structural equation modeling technique. A second hypothesis was that fatigue would also influence change in depression over time but at a lesser magnitude. Depression and fatigue were strongly correlated in the sample (i.e., baseline correlation of latent variables was 0.72). Although the model showed good fit to the data, χ2 (66, N = 329) = 88.16, p = 0.04, SRMR = 0.030, RMSEA = 0.032, and CFI = 1, neither cross-lagged structural path was significant. The findings suggest that depression had no causal influence on changes in fatigue in this sample, and fatigue did not influence change in depression. The clinical implication is that depression treatment may not be helpful as a treatment for CRF and therefore interventions specifically targeting fatigue may be needed. Future research should include additional waves of data and larger sample sizes.

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73α, and E2F1 to the hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer.

Further studies of Stk31 in spermatogenesis in vivo would allow the identification of the asymmetry machinery of GSCs and the signaling mechanism underlying cell fate determination. Further studies of STK31 in cancer stem cells would allow the development of new diagnostic and therapeutic approaches. / In the first part of the experiment, the expression and cellular localization of STK31 were investigated. RT-PCR results showed that STK31 was reactivated in 47 -- 86% of multiple cancers. Immunofluorescent study and GFP tagging experiment showed that STK31 was localized in the cytoplasm and formed aggregated granules that divide asymmetrically during mitosis. Further study by co-staining with E-cadherin demonstrated that the mouse homolog, Stk31, was expressed in the transition state between undifferentiated and differentiated spermatogonia. These data suggest the possible involvement of STK31 in mouse spermatogonia and cancer development. / In the second part of the experiment, the function of Stk31 in mouse spermatogonia was investigated- A GSC culture on an STO feeder layer was established. Studies on growing properties, expression of molecular markers and germ cell transplantation showed that GSC culture maintained spermatogonial stem cell activity. Retinoic acid was then used to induce differentiation of GSC. The differentiation status was confirmed by monitoring the expression of molecular markers. RT-PCR and immunofluorescent study showed that the expression of Stk31 was induced in RA-induced differentiation and Stk31 proteins were asymmetrically distributed during GSC division. Overexpression of Stk31 in GSCs using retroviral transduction induced the differentiation phenotypes. These data indicate the involvement of Stk31 in mouse spermatogonia cell fate determination. / In the third part of the experiment, the function of STK31 in human colon cancer was investigated. A stable STK31 knock-down Caco2 cells were established by stably transfecting two miR RNAi designs with different efficiency into Caco2 cells. Flow cytometry analysis showed that knock-down of STK31 resulted in G1 phase arrest. Cell counts and MTS assays suggested that knock-down of STK31 decreased cell proliferation in confluent cultures. Knock-down of STK31 also enhanced cell attachment to several ECM proteins and decreases cell migration as suggested by attachment assays and migration assays. Moreover, knock-down of STK31 enhanced enterocytic differentiation and inhibited tumorigenicity both in vitro and in vivo as indicated by colony formation assays and xenograft assays. Date obtained from whole genome microarray studies indicate that STK31 regulates these "stemness" properties through altering the expression of key players in various pathways including KIT, SMAD1 and Cyclin D2. These results suggest the involvement of STK31 in colon cancer as a regulator of "sternness". / Spermatogenesis is a complicated process involving mitosis, meiosis and post-meiotic differentiation. Due to the lack of in vitro models, genes that are involved in mammalian spermatogenesis are largely unknown. Spermatogenesis and tumorigenesis share important biological similarities. This co-relation can be signified by a special group of genes called cancer/testis (CT) antigens, which are only expressed in the testes and cancer. Although cancer biology has been extensively studied for decades, promising therapeutic methods are not available for every type of cancer. Recent discovery of cancer stem cells and functional genomics studies have shed light on the development of new diagnostic and therapeutic approaches. This thesis describes the expression, cellular localization and function of a novel CT gene, STK31, in spermatogonia and cancer development. / Fok, Kin Lam Ellis. / "December 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 143-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.