Glioblastoma is the most common malignant brain tumor, accounting for 17% of all primary brain tumors in the United States. Despite the available surgical, radiation, and chemical therapeutic options, the invasive and infiltrative nature of the tumor render current treatment options minimally effective. Recent reports have identified multiple regulators of glioblastoma progression and invasiveness. It has been demonstrated that ADAM12, A Disintegrin And Metalloproteinase encoded by ADAM12 gene, is over-expressed in glioblastoma and directly correlated with tumor proliferation. Additionally, dysregulation of the Notch signaling pathway has been implicated in the pathogenesis of many gliomas. Lastly, an evolving role of microRNAs, small noncoding RNAs, in carcinogenesis is progressively growing. A recent study has identified ADAM12 as a notch-related gene, and another demonstrated that inhibition of notch signaling decreased glioblastoma recurrence. However the mechanisms of regulation are still unknown. In this study, we hypothesize that direct downregulation of microRNA-29, downstream of over-expression of notch enhances glioblastoma malignancy through upregulation of ADAM12. Although our data demonstrate upregulation of Notch1, its downstream target HES1, and ADAM12 in U87MG glioblastoma cell line. Expression of the cleaved intracellular Notch1 was not detected. Furthermore, we were unable to demonstrate an inhibitory effect of ɣ-secretase inhibitor on Notch signaling, likely reflecting the requirement for modifying culturing conditions or detection in our assays. Furthermore, miR-29 was detected in glioblastoma cells. The expression of miR-29 was further elevated by ɣ-secretase inhibitor treatment, suggesting a role for Notch1 inhibition on miR-29 expression. Although no conclusive results are shown in our work, a role of Notch1 through miR-29 is implicated in the pathogenesis of glioblastoma pathogenesis warranting further investigation into the role downstream target genes in the Notch signaling pathway.

Intratumor heterogeneity poses a significant challenge for the diagnosis and treatment of patients with breast cancer because distinct sub-populations of tumor cells contribute significantly more to therapy resistance and tumor recurrence than others. Consequently, understanding the mechanisms that contribute to this heterogeneity and identifying sub-populations responsible for aggressive behavior is a significant and timely problem. Considerable evidence indicates that a subpopulation of tumor cells with stem/progenitor-like characteristics, termed cancer stem cells (CSCs), is responsible for therapy resistance and recurrence, sparking interest in characterizing novel biomarkers and therapeutic targets for this aggressive population of cells. Unfortunately, CSCs share many protein markers with normal mammary stem/progenitor populations, minimizing potential targets for diagnostic and therapeutic purposes. Therefore, in my thesis research, I investigated novel ways to identify CSC populations based on their glycome. I observed that breast CSCs have a unique glycosylation pattern that can be used to distinguish them from other tumor populations. Specifically, I discovered a novel α2,3 sialoglycan on Core2 O-linked glycans expressed on CSCs that can identified using the lectin SLBR-N. I found that SLBR-N can be used to distinguish CSCs from bulk tumor cells in multiple in vitro and in vivo models. I also discovered that the CSC marker, CD44s, expresses O-linked α2,3 sialoglycan and that this glycan alters CD44s function by promoting the activation of the PDGFRβ/STAT3 pathway. In contrast, the fucosyltransferase FUT3 and its glycan sialyl Lewis X (sLeX) are expressed on non-CSCs and they function to impede stemness by inhibiting CD44s-mediated PDGFRβ/STAT3 signaling. In summary, this thesis provides insights into glycan heterogeneity in breast cancer and novel ways to identify CSCs using the glycome.

Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts with NER factor xeroderma pigmentosum group A (XPA) where an ATR phosphorylation site on serine 196 is located. Phosphorylation of XPA on serine 196 is required for repair of UV-induced DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the UV-induced XPA phosphorylation and DNA repair. Moreover, we show that depletion of p53, a downstream checkpoint of ATR, and inhibition of p53 transcriptional activities reduced the UV-induced XPA import. Furthermore, we found that the ATR-directed XPA nuclear import happens primarily in the S phase of the cell cycle. In effort to determine the mechanism involved in the XPA nuclear import, we found that, in addition to the nuclear localization signal (NLS) of XPA, importin-α4 is required for the UV-induced XPA nuclear import in an ATR-dependent manner. These data suggest that NER could be regulated by the ATR-dependent checkpoint via modulation of XPA phosphorylation and nuclear import. In a separate study we show that, upon UV damage, cytoplasmic ATR translocates to mitochondria, blocks the recruitment of proapoptotic Bcl-2–associated X (Bax) protein to mitochondria and prevents the loss of mitochondrial membrane potential (ΔΨ) and apoptosis. Bax-depletion reduces the effect of ATR on ΔΨ. Remarkably, the cytoplasmic ATR exhibits no checkpoint kinase activity, a hallmark function of nuclear ATR. Silencing of ATR’s kinase activity failed to affect Bax relocalization to mitochondria. These results reveal a novel checkpoint-independent antiapoptotic function of ATR at mitochondria in the cellular response to DNA damage.

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation of the epidermal growth factor receptor (EGFR). As a result, the cellular response to EGF was reduced as shown by the down-regulation of EGF-stimulated Erk1/2 phosphorylation. The expression of Slug, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and granulocyte-macrophage colony stimulating factor (GM-CSF) was also down-regulated by prostasin re-expression in the PC-3 cells. Co-expression of prostasin and its activating protease matriptase with EGFR in FT-293 cells induces an apparent proteolytic cleavage of the EGFR in the extracellular domain at two specific sites, generating two N-terminally truncated EGFR fragments, named EGFR135 and EGFR110. The EGFR110 is constitutively tyrosine-phosphorylated, and in its presence the phosphorylation of downstream signaling molecules including Erk1/2 and Akt is increased under serum-free conditions. Neither EGFR135 nor EGFR110 is responsive to EGF stimulation. Deletions of the EGFR extracellular domain (ECD) were generated to map the matriptase-prostasin cleavage sites. Two candidate sites were localized to regions AA1-273 and AA273-410. These data support a mechanism of action for the matriptase-prostasin epithelial extracellular serine protease activation cascade by proteolytically modulating the EGF-EGFR signaling. Prostasin gene expression is down-regulated in high-grade and hormone-refractory prostate cancers. We investigated the mechanisms by which androgens regulate prostasin expression in the prostate and prostate cancer. We treated the LNCaP human prostate cancer cells with dihydrotestosterone (DHT) and measured the mRNA expression of prostasin and potential transcription regulators of prostasin predicted by interrogation of the prostasin gene promoter sequence. Prostasin mRNA expression in the LNCaP cells was not responsive to DHT treatment. DHT marginally up-regulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of a prostasin promoter-reporter and SREBP cDNA in HEK-293 cells resulted in stimulation of the promoter activity at ~2 fold by SREBP-1c, and up to 6 fold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of the promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2 s stimulation of the prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 cells resulted in up-regulation of the endogenously expressed prostasin while transfection of a SLUG cDNA in the LNCaP cells repressed prostasin expression. Multiple SREBP-2 binding sites, known as sterol regulatory elements (SRE s), were identified at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Mutagenesis of the five SRE s was carried out to evaluate their roles in SREBP-2 up-regulation of prostasin. SRE+98, a novel functional sterol regulatory element was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. CONCLUSIONS: Prostasin regulates the expression of several invasion-promoting molecules in prostate cancer cells by down-modulating the EGF-EGFR signaling pathway. Active prostasin induces proteolytic cleavage in the EGFR ECD at two specific sites. One of the N-terminally truncated EGFR, the EGFR110 is auto-phosphorylated along with increased phosphorylation of downstream signaling molecules. The effect of the androgen DHT on prostasin expression in prostate cells is mediated via SREBP s, which stimulate the promoter, and Slug, which represses the promoter. Slug is up-regulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

LIM kinase 1 (LIMK1) is a unique dual specificity serine/threonine kinase containing two N-terminal LIM domains in tandem, a PDZ domain and a C-terminal catalytic domain. LIMK1 is involved in modulation of actin cytoskeleton through inactivating phosphorylation of the ADF (actin depolymerization factor) family protein cofilin. Recent studies have shown that LIMK1 is upregulated in breast and prostate cancer cells and tissues, promotes metastasis in animals and induces acquisition of an invasive phenotype when ectopically expressed in benign prostate epithelial (BPH) cells. Furthermore, overexpression of LIMK1 was associated with altered sub cellular localization of the membrane type 1 matrix metalloprotease (MT1-MMP). Matrix metalloproteases (MMPs) are a family of zinc dependant proteolytic enzymes that hydrolyze extra cellular matrix and cell surface molecules. A number of MMPs including MMP-2, MMP-9 and their activator MT1-MMP are over expressed in a variety of cancers including prostate cancer. The abundant expression of these enzymes contributes to changes in the tumor microenvironment, which facilitate degradation of the surrounding collagen matrix and migration of cells through the matrix defects. In this study, we show that MMPs are involved in LIMK1 induced invasion of otherwise non-invasive BPH cells. We also show that (a) the kinase activity of LIMK is not essential for the invasive behavior of the cells and (b) the absence of LIM domains significantly retards cell invasion. We have established transfected sub lines of BPH cells stably expressing 1) constitutively active LIMK1 (BPHLCA), 2) kinase dead LIMK1 (BPHLKD) and 3) only the kinase domain of LIMK1 (BPHLK) for our study. In vitro invasion assays revealed that LIMK1 induced invasion was inhibited by the MMP specific inhibitor, GM6001, and that cells expressing kinase-dead LIMK1 were equally invasive. Furthermore, BPH cells expressing LIMK1 mutants expressed higher amounts of MMP-2 and MMP-9. Substrate zymography revealed increased concentration of secreted MMP-2 and MMP-9 in the media of BPHLCA and BPHLK cells respectively compared to BPHV (vector control) cells. Quantitative RT-PCR also showed a ~10 fold increase in the steady state concentration of MMP-2 in BPHLCA cells compared to the control BPHLV cells. Expression of active LIMK1 stimulated cell-surface expression of MT1-MMP in BPHLCA cells as determined by flow cytometry. A modest increase in expression of MT1-MMP was noted in BPHLKD cells compared to BPHLK and BPHV cells. Immunoflourescence analysis indicated differential localization of MT1-MMP and LIMK1 in BPH cells expressing different mutants of LIMK1. Co-localization of LIMK1 and MT1-MMP in the plasma membrane and in the perinuclear region was also evident in these cells. Furthermore, here we provide evidence that suggests a functional role for phosphorylated (activated) LIMK1/2 (p-LIMK1/2) during mitosis through its association with γ-tubulin. Immunoflourescence analysis showed distinct co-localization of γ -tubulin and p-LIMK1/2 in the centrosomes during mitosis from early prophase to the beginning of telophase. No association was seen in the interphase or in late telophase. Phospho-LIMK1/2 was co-precipitated in immunoprecipitates of γ -tubulin using an anti- γ -tubulin antibody suggesting a physical association between these proteins in a complex. This finding reveals a novel role of LIMK1 in the mitotic process. In summary, our data suggests that MMPs are involved in LIMK1 induced invasion of prostate epithelial cells, and that this effect is mediated through altered expression and activation of specific MMPs. Furthermore, LIMK1 induced invasion is dependant on the presence of LIM domains more than the kinase activity. Finally, we show that phosphorylated LIMK1 and LIMK2 are involved in the mitotic process in a stage specific manner through its association with the centrosomal protein γ -tubulin. Because LIMK1 promotes invasion in vitro, regulates expression of MMPs, and is involved in mitotic processes, it is an attractive drug target for prostate cancer therapy.

Ultraviolet (UV) radiation has been demonstrated in numerous studies to be a major risk factor for non-melanoma skin cancer development. Despite the emergence of current UV-preventative strategies, such as sunscreens and skin-protective clothing, the incidence of non-melanoma skin cancer has continued to rise. This has encouraged investigations on alternative methods for UV prevention. In particular, bovine milk sphingomyelin has been studied for its potential in protecting human skin against UV photodamage. While the previous studies have suggested that sphingomyelin exhibits UV-protective properties in a human skin equivalent model, the exact mechanisms behind sphingomyelin’s photoprotective effects are yet unknown. This thesis aims to further investigate the UV-protective effects of sphingomyelin in normal human epidermal keratinocytes, using nuclear p21 expression as a marker for UV photodamage. Keratinocytes were incubated for 24 hours in a 0.1% sphingomyelin solution and then exposed to 40mJ/cm2 of 302nm UV radiation. After 24 hours of post-UV incubation, nuclear p21 expression was evaluated using immunofluorescence. Confocal images were analyzed for their mean nuclear p21 fluorescence intensity measured in grayscale (0-255). Keratinocytes treated with sphingomyelin showed approximately a 50% decrease in UV-induced mean nuclear p21 intensity compared to keratinocytes with no sphingomyelin treatment (via Tukey’s test; p

For the past 40 years, antiangiogenic approaches have been of major interest in the development of methods to cure and prevent cancer. Angiogenesis, the development of blood vessels from pre-existing vascularization, is essential for cancer growth and spread of metastasis through the delivery of nutrients and oxygen essential to sustain the metabolic activity of these malignant cells. Blocking access to blood will cause cancerous cells to assume a dormant state creating inactive micro-tumors innocuous to the host. Angiostatin, the internal fragment of the fibrinolytic zymogen plasminogen, has shown great potential in reducing cancer size and number of metastatic colonies in animal models. Owing to the success of these preliminary results angiostatin is currently on clinical trials. Plasminogen is known to be transferred from blood to milk during lactation. The objectives of this research were to: 1) investigate the ability of various proteases in cleaving plasminogen, both from human and bovine sources, and consequently release the angiostatin like fragment; 2) determine the anticancer activity of bovine angiostatin; 3) examine ability of the antiangiogenic fragment to survive digestion; 4) purify the fragment of interest through column chromatography. Production of angiostatin was tested through hydrolysis of plasminogen via Bacillus Polymyxa protease (or dispase I), elastase, lactic acid bacteria and Bacilli originated enzymes. Once proteases capable of angiostatin like peptide production were identified, and sequence analysis of the fragments obtained conducted to confirm that bovine angiostatin was indeed produced, ability of angiostatin, both human and bovine, in inhibiting malignant melanoma as well as colon cancer cells was evaluated in vitro. From the results obtained we can confirm that bovine angiostatin inhibitory activity on cancerous cells is similar to that observed for human angiostatin. Analysis of bovine angiostatin survival through in vitro human digestion model was also examined. Results show good possibility of angiostatin surviving digestion, even if confirmation of these results is required through further in vivo studies. Additionally, digestive enzymes such as trypsin and α-chymotrypsin showed ability in cleaving plasminogen directly to release a 25kDa fragment. Knowing that each kringle has some degree of anticancer activity it would be of interest to further study the possibility of angiostatin related fragments to be produced during milk digestion. Finally, affinity chromatography through L-lysine used to purify human angiostatin resulted to be an adequate method for bovine angiostatin purification. Preliminary results obtained from this study open a new area worth investigating to uncover the potential of using bovine angiostatin in the development of novel food products capable of cancer prevention.

Fusion of cancer cells has been observed in tumors for more than a century and is thought to contribute to tumor development and drug resistance. The low frequency of cell fusion events and the instability of fused cells have hindered our ability to understand the molecular mechanisms that govern cell fusion. We have developed a patterned gel system that can isolate cell fusion events and we demonstrate that several breast cancer cell lines can fuse into multinucleated giant cells in vitro, and the initiation and longevity of fused cells can be regulated solely by biophysical factors. Dynamically tuning the adhesive area of the micropatterned substrates, reducing cytoskeletal tensions pharmacologically, altering matrix stiffness, and modulating pattern curvature all supported the spontaneous fusion and stability of these multinucleated giant cells. These observations highlight that the biomechanical microenvironment of cancer cells, including the matrix rigidity and interfacial curvature, can directly modulate their fusogenicity, an unexplored mechanism through which biophysical cues regulate tumor progression.

The CpG Island Methylator Phenotype (CIMP) is a distinct form of aberrant DNA methylation in cancer, and it is seen in 20-40% of colorectal cancers (CRC) where its causes remain elusive. Intestinal microbiota represents an important environmental component implicated in CRC development. Interestingly, microbiota have been shown to modulate DNA methylation in preclinical models but the relationship between tumor-infiltrating bacteria and CIMP status in currently unknown. Our hypothesis is that the gut microbiota affects colonic neoplasia through modulating aberrant DNA methylation in host epigenome. To test this hypothesis, we analyzed CIMP status in CRC patient tumor samples. We used a genome-wide approach to determine the CIMP status by filtering cancer-related sites. A total of 1317 CpG sites were filtered and used to determine distinct CIMP classifications that aligned with well-known characteristics of CIMP cases, including localization in the proximal colon, a higher prevalence in female patients, and a higher frequency of MLH1 hypermethylation. To study the association between CIMP and the gut microbiota, we analyzed the enrichment of four bacterial species associated with CRC, including Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae. Notably, they exhibited higher enrichment in CIMP-Positive tumor samples, except for E. coli. This analysis also identified a group of samples referred to as bacterial "Superhigh," characterized by remarkably high abundances of these three bacterial species. The bacterial Superhigh cases displayed a significant association with CIMP status and MLH1 methylation. We validated the association between the CRC-associated bacteria and CIMP by analyzing the Cancer Genome Atlas (TCGA) 450K methylation array data and whole exome sequencing data. The analysis demonstrated that bacterial Superhigh cases in the TCGA datasets also had significantly higher odds of being CIMP-Positive and having MLH1 methylation. To expand our investigation, we conducted 16S rRNA gene sequencing to identify additional bacterial taxa linked to CIMP. Numerous bacterial genera and species were found to be enriched in CRC tumor tissues, with specific enrichments in CIMP-Positive and CIMP-High groups. Notably, Bergeyella, Campylobacter concisus, and Fusobacterium canifelinum were significantly enriched in CIMP-Positive tumors. Additionally, I studied the causal relationship between gut microbiota and CpG island methylation by colonizing germ-free mice ApcMinΔ850/+;Il10–/– with E. coli NC101 & K. pneumoniae, specific pathogen free bacteria, and the mouse bacterial Consortium. Differential methylation analyses of adjacent normal colon tissue revealed a pronounced tissue side-specific difference, particularly in non-CpG island regions. The tissue specificity diminished with the increasing tumorigenic potential of the microbiota group. Comparisons between microbiota groups and germ-free mice indicated a more significant increase in methylation within CpG islands when gut microbiota with higher tumorigenic potential was present. In conclusion, our study underscores the association between CIMP in CRC and the gut microbiota and the causal relationship between the cut microbiota and CpG island methylation. It highlights specific bacterial taxa that may impact DNA methylation especially in CpG islands and contribute to the development ang progression of CIMP in colorectal cancer. / Biomedical Sciences

Polyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-α-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport. Thus, there is a need to develop drugs that inhibit polyamine transport to use in combination with DFMO. Surprisingly, little is known about the polyamine transport system in humans and other eukaryotes. Understanding the transport system would allow us to identify compounds that inhibit polyamine transport, which could then be used in tandem with DFMO to treat cancer. Our laboratory has identified one gene in Drosophila, called CG32000, as a component of this transport system, and numerous other candidate genes remain to be tested. To better characterize this system, this project investigated the ability of the Drosophila transport system to take up a toxic polyamine analogue called norspermidine, with the initial goal of developing a new screening method to find polyamine transport genes. My experiments have demonstrated significant differences in norspermidine uptake and toxicity between C. elegans and Drosophila which may imply a secondary polyamine transport system in higher eukaryotes. In the long term, it is hoped that this thesis will facilitate the development of more effective cancer medications by providing new information about the polyamine transport system.