Non alcoholic fatty liver disease (NAFLD), is recognized as the one of the most common causes of liver disease in the United States and worldwide. NAFLD is associated with increased risk of development of hepatocellular carcinoma. Additionally, our lab has shown an increased metastatic burden in the steatotic liver using a mouse model of diet induced steatosis. NAFLD is characterized by cellular and molecular changes in the liver which include an influx of inflammatory cells, changes in gene expression and alteration in cytokine production. We hypothesize that changes in the steatotic liver contribute to a more permissive microenvironment for tumor growth and establishment of metastases. To better understand the alterations to the liver with NAFLD, we used a murine model of steatosis and corroborated our results with human samples of NAFLD. Analysis of inflammatory cell populations revealed increased infiltration of CD11b positive myeloid and CD3 positive lymphocytic cell populations in steatotic livers compared to normal livers. Significant alterations in cytokine profiles in the plasma and liver tissue lysates from normal and steatotic mice were detected including leptin, CXCL1, CXCL2, and CXCL16 that were further shown to directly increase hepatocyte proliferation in vitro. To determine molecular factors altered with NAFLD, we assessed changes in matrix metalloproteinase levels and show that MMP13, an interstitial collagenase, is significantly upregulated in the steatotic liver. To evaluate the role of host MMP13 on tumor development, we used the splenic injection model of liver metastasis in mice genetically deficient in MMP13 and show a significant decrease in metastatic tumor burden in MMP13-/- mice compared to wildtype mice. Using confocal microscopy we observed a significant decrease in the number of individual tumor cells extravasating from the hepatic vasculature in MMP13-/- mice compared to wildtype mice. Stable MMP13 knockdown cell lines were used to demonstrate that reduced tumor derived MMP13 decreased migratory and invasive properties in vitro and decreased metastatic burden in vivo. This study identifies changes in the steatotic liver that impact tumor development and establishment of metastases in the steatotic liver microenvironment.

Stromal-epithelial interactions are important in both prostate development and cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. Several molecules found to be aberrantly expressed in cancer associated fibroblasts (CAFs) (including cyclin D1 [CD1], stromal derived factor 1 [SDF-1]) contribute to tumorigenesis and malignant transformation in xenograft experiments. These molecules can be regulated by a number of different factors, but are both putative estrogen regulated genes. In this study, we show that dysregulation of ERα expression in cancer associated stroma results in the differential regulation of estrogen responsive genes that are key factors in enhancing the invasive potential of the epithelial tumor. The cell cycle regulator CD1 and the estrogen receptor are known to interact and can induce estrogenic gene transcription. Cathepsin D (CathD) is an estrogen regulated aspartic endopeptidase, known to be involved in a number of physiological processes as well as in the regulation of apoptosis. In this study, we highlight CathD as a mediator of cancer associated stromal promotion of prostate tumorigenesis. An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue in comparison to benign prostate tissue. Stromal specific overexpression of CathD in benign prostate stromal cells induced malignancy in adjacent epithelium through increased TGFβ signaling and responsive gene expression. The proteolytic function of stromally-derived CathD is dependent on the activity of hydrogen-proton pump activity on the surface of prostate epithelial cell lines. The study presented here indicates that CathD is not only an important mediator of stroma-epithelial cross talk, but also an essential component in promotion of tumorigenesis in vivo, and Inhibition of ER signaling in the cancer associated stroma inhibits malignant transformation in the adjacent epithelium.

The calcium activated chloride regulator hCLCA2 (human, calcium activated chloride channel) is thought to be a breast tumor suppressor. Its mouse homolog mCLCA5 is upregulated during mammary involution and in vitro by withdrawal of growth factors, cell detachment, and contact inhibition. Cell to matrix adhesion is a key factor involved in cellular homeostasis and disruption of such interaction induces a special kind of apoptosis called anoikis (homelessness). The cell has to bypass anchorage dependent growth in particular during metastasis as well as wound healing. Detachment-induced apoptosis, or anoikis, is one of the primary obstacles to metastasis. We have observed that human CLCA2 is abundantly expressed in normal mammary epithelial cells but downregulated with tumor progression as cells become anoikis-resistant. We show that re-introduction of hCLCA2 into breast cancer cell lines induces apoptosis both in anoikis-sensitive and anoikis-resistant breast cancer cell lines. In immortalized cells, there are increases in p53 and the activated form of ERK (Extracellular Receptor Kinase) prior to cell death. Furthermore, hCLCA2 expression is up-regulated by serum withdrawal and cell detachment in mammary epithelial cell lines. Taken together, these results suggest an important role for hCLCA2 in anoikis. Cell detachment or serum withdrawal inactivates the PI3K-Akt pathway, allowing the Akt target FOXO transcription factors to enter the nucleus and transactivate genes responsible for cell cycle arrest and apoptosis. Here we show that inhibiting the PI3K-Akt pathway either with LY294002 or wortmannin induces hCLCA2 expression in MCF-7 and MDA-MB-231 breast cancer cells. We further show that adenoviruses encoding FOXO3A and dominant-negative Akt induce hCLCA2 transcription in breast cancer cell lines. hCLCA2 is upregulated only by FOXO3A not by FOXO1A as shown by luciferase reporter assays. The hCLCA2 promoter contains a potential binding site for FOXO3A within 300bp upstream of the transcription start site. Deletion analysis of the hCLCA2 promoter using luciferase fusions confirms that a FOXO3A-responsive site lies within that interval. Moreover, the upregulation of hCLCA2 by FOXO3A is dependent upon p53, a binding partner of FOXO3A. This was confirmed by luciferase reporter assays using a FOXO3A-DNA binding mutant and p53 knockdown. Finally, ectopic expression of hCLCA2 sensitized MCF7 cells to anoikis. These results demonstrate that hCLCA2 is upregulated upon cell detachment or serum withdrawal through the PI3K-Akt-FOXO3A pathway and plays a role in cell detachment mediated apoptosis.

Metastasis persists as a significant unsolved hurdle in cancer treatment, with greater than 90% of cancer-related deaths attributed to metastasis. In order for cells to successfully metastasize, they must dynamically regulate cell adhesion. However, cell adhesion molecules are rarely mutated or deleted genetically in cancer, indicating that tumor cells are able to co-opt intrinsic regulatory mechanisms of adhesion to drive metastasis. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that tunable regulation of its function contributes to tumor cell adhesion and metastasis. We tested this hypothesis through two channels. We identified ALCAM as a novel binding partner of tetraspanin CD151, a known regulator of cell adhesion and motility. We previously demonstrated that clustering of integrin-free CD151 (CD151free) increased cell adhesion, decreased cell motility, and inhibited metastasis. Here, we identified ALCAM as a novel CD151 partner required for CD151free to control adhesion. Biochemical analyses revealed that CD151free is coupled to ALCAM by the scaffolding protein syntenin-1. Additionally, we show that the intracellular domain of ALCAM (ALCAM-ICD) is susceptible to ɣ-secretase cleavage, which releases a PDZ-binding peptide capable of disrupting the CD151/syntenin-1/ALCAM complex. Disruption of this complex impedes CD151free-mediated regulation of tumor cell adhesion and metastasis, demonstrating that CD151free controls tumor cell migration through a trimeric complex of CD151/syntenin-1/ALCAM. Further evaluate of ALCAM revealed a potential alternative splicing which we predicted to control proteolytic shedding of its extracellular domain. We demonstrate that the loss of the membrane-proximal exon13 generates an ALCAM splice variant (ALCAM-Iso2) that enhances metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent, membrane distal cleavage site in ALCAM-Iso2, which increases shedding ten-fold, thereby decreasing cellular cohesion and promoting motility. ALCAM-Iso2-expression was greatly increased in bladder cancer, further emphasizing that ALCAM alternative splicing can contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of dissemination is a locally tunable process. In summary, this dissertation presents two mechanisms by which tumor cells are able to dynamically regulate cell adhesion to modulate migration and metastasis.

Cell motility is a process tightly linked to tumor metastasis. The p21-activated kinases (PAKs) are modulators of cell motility commonly overexpressed and hyperactive in various malignancies. We investigated whether increased PAK2 expression and phosphorylation levels enhance cancer cell motility, thus worsening disease prognosis in non-small cell lung cancer (NSCLC). In silico analyses of publically available databases was accessed to determine the clinical relevance of PAK2 gene expression in NSCLC patient outcomes. Total and phospho-specific PAK1 and PAK2 antibodies were used to screen a panel of NSCLC cell lines. shRNA and small molecule inhibitors were utilized to assess the role of PAK1/2 kinases in augmenting tumor metastatic capacity in vitro by two and three-dimensional cell migration assays. Increased tumor PAK2 expression was associated with significantly worse survival in two unique cohorts of patients with resected lung adenocarcinoma. Phosphorylated PAK1/2 isoforms were frequently but variably expressed across 31 NSCLC cell lines, and at higher levels than in immortalized normal bronchial epithelial cells. We observed a dependency on PAK2 for cell motility specifically in cell lines with phosphorylated PAK1/2. Additionally, use of two PAK small molecule inhibitors recapitulated the effects observed in our PAK2 shRNA mediated knockdown studies. Our findings demonstrate the importance of PAK2 signaling in promoting lung cancer cell motility and are the first to link high PAK2 expression to poor clinical prognosis in lung adenocarcinoma. These data suggest that targeting PAK2 may cause an anti-migratory effect and potentially improve survival for NSCLC patients with elevated tumor PAK2 expression levels.

Ewing sarcoma is a highly aggressive pediatric bone tumor that is characterized EWS/FLI1 transcription factor. Ewing sarcoma tumors require the continued activity of EWS/FLI1 to sustain a gene signature that promotes proliferation and blocks differentiation leading to tumorigenesis and disease progression. Inhibition of EWS/FLI1 activity is not compatible with cell proliferation, which creates an attractive drug target. While no EWS/FLI1 targeted therapies have been translated into the clinic, trabectedin has demonstrated activity in early phase clinical trials. In this report, we optimize trabectedin therapy for the treatment of Ewing sarcoma. We identify the mechanism by which trabectedin inhibits EWS/FLI1 activity and demonstrate that inhibition can be achieved at clinically relevant concentrations. We use this novel mechanism of action to further optimize the schedule of administration and show that maximum EWS/FLI1 inhibition is obtained following a short term, high concentration treatment with trabectedin. Additionally, we characterize a second-generation analog of trabectedin, lurbinectedin, which has an improved pharmacokinetic profile that allows much higher serum concentrations to be achieved. In addition to being a bonafide EWS/FLI1 inhibitor, lurbinectedin can be combined with SN38 (in vitro) or irinotecan (in vivo) to augment the suppression of EWS/FLI1 target genes. Ultimately, we show that this combination strategy decreases tumor growth, extends lifespan, and leads to the differentiation of xenograft mouse models of Ewing sarcoma.

Evasion of cell death is essential to every step of tumorigenesis. Anti-apoptotic Bcl-2 family proteins are master inhibitors of cell death, and thus are often overexpressed in cancers. In particular, Bcl-2, Bcl-xL, and Mcl-1 are highly expressed in Estrogen Receptor-alpha positive (ERα+) breast cancers. However, one anti-apoptotic Bcl-2 family protein, Mcl-1, is understudied in ERα+ breast cancers. Herein we show that Mcl-1 is essential to ERα+ breast tumor cell survival, and is a more potent tumor cell survival factor than other family members, like Bcl-2 and Bcl-xL. Interestingly, Mcl-1 expression and activity increased upon Bcl-2/Bcl-xL dual inhibition with ABT-263, mediated by increased Mcl-1 cap-dependent translation. Blockade of cap-dependent translation, through inhibition of mTORC1 signaling, resulted in tumor cell killing in cell culture and in vivo, phenocopying ablation of Mcl-1 by RNA-interference. Mcl-1 depletion in combination with ABT-263 restored sensitivity to Bcl-2/Bcl-xL blockade, suggesting that Mcl-1 is a primary resistance factor to Bcl-2/Bcl-xL inhibition in ERα+ breast cancers. Importantly, preliminary studies suggest that anti-apoptotic Bcl-2 family proteins can promote resistance to standard of care breast cancer therapies. Mcl-1 inhibition, but not Bcl-2/Bcl-xL blockade, promoted cell death in a model of anti-estrogen resistance, long term estrogen deprivation (LTED). Mcl-1 inhibition using polymeric nanoparticles containing Mcl-1 siRNA (si-NPs), increased tumor cell death in combination with LTED and after treatment with the selective estrogen receptor downregulator fulvestrant. Therefore, Mcl-1 is a dominant tumor cell survival factor in ERα+ breast cancers that is rapidly and potently upregulated in response to targeted therapies. However, dependence on Mcl-1 for tumor cell survival may be clinically thwarted using mTOR inhibitors or si-NPs.

The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions caused by breast tumors, patients still die of the disease, and the current treatments for these lesions are palliative. Thus, there is a need to identify the early determinants of the breast cancer bone metastatic process in order to treat these lesions more effectively. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. Interestingly, the bone is innervated by nerves of the sympathetic nervous system (SNS), and previous data from our lab show that chronic SNS stimulation promotes breast cancer colonization of bone. What has not been explored is how chronic SNS activity alters the bone vasculature, a key component in successful osteotropic breast cancer metastases. The research presented in this dissertation shows that stimulation of the beta 2-adrenergic receptors (b2AR) specifically on osteoblasts alters bone vascular density and the adhesion protein profile of bone marrow endothelium via increased osetoblastic Vegf-a and Il-6. These results support the notion that chronic stress affects not only colonization of bone by breast cancer cells, but also the interaction between endothelium and tumor cells that occurs during extravasation. Potential clinical benefits of beta-blockers, anti-angiogenics, and inhibitors that target adhesion need to be investigated further regarding breast cancer bone metastases.

The native polyamines putrescine, spermidine, and spermine are small positively-charged molecules that can interact with negatively-charged macromolecules like DNA, RNA and proteins. These interactions aid in nucleic acid and protein conformational stability, thereby, making polyamines essential building blocks for cells. Polyamines are involved in various cellular functions including gene regulation, protein synthesis, and cell proliferation. Rapidly-proliferating cells, such as cancer cells, utilize high polyamine levels for cell growth. Targeting the polyamine addiction of cancers is a validated anti-cancer strategy. To achieve the maximal reduction of polyamine levels, polyamine blocking therapy (PBT) employs several compounds in combination. First, one can inhibit polyamine biosynthesis using difluoromethylornithine (DFMO). DFMO inhibits ornithine decarboxylase (ODC); a rate limiting enzyme required for the generation of putrescine. Unfortunately, inhibition of polyamine biosynthesis is often insufficient because cells escape DFMO pressure by upregulating polyamine import. To address this escape pathway, a polyamine transport inhibitor (PTI) is included with DFMO to reduce cell growth via PBT. There are two types of PTIs: polyamine-based and non-polyamine-based PTIs. Chapter 1 provides an overview of the field. In Chapter 2, we developed a novel polyamine-based PTI which is smaller and less toxic than the known PTIs and was equally potent when used in PBT. In Chapter 3, we identified the first non-polyamine-based, PTI (GW5074) that successfully reduced human cancer cell growth when dosed with DFMO. In Chapter 4, we discovered new DFMO adjunct agents which inhibit the far upstream element binding protein 1 (FUBP1). This type of PBT significantly reduced cancer cell growth and was directed at polyamine biosynthesis via a two-fold mechanism involving direct inhibition of polyamine biosynthesis (DFMO) and downregulation of upstream transcription factors like c-myc. In summary, we have described three categories of compounds that can be used as adjunctive agents for DFMO in anti-cancer therapies.

Neuroblastoma is an aggressive pediatric cancer that is poorly responsive to traditional cancer therapies. Oncolytic viral (OV) vectors such as Zika virus (ZIKV) and Parainfluenza virus type 5 (P/V virus) are promising neuroblastoma therapeutics, but the role of innate immune responses in the effectiveness of OV killing is not well understood. Previous studies showed the neuroblastoma cell line SK-N-AS (expressing low CD24) had low permissivity for ZIKV infection, and this restriction was relieved by ectopic CD24 expression (CD24-high cells). Compared to permissive CD24-high cells, the non-permissive CD24-low cells had elevated basal levels of IRF-1, NF?B and phosphorylated STAT1; these cells also showed higher levels of interferon (IFN)-induced antiviral genes and activity against IFN- sensitive viruses (e.g., VSV). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor. Transcriptomics analysis revealed that CD24 expression suppressed basal expression of antiviral genes. These data support the proposal that CD24 alters basal and induced antiviral states and may represent a novel biomarker for susceptibility of cells to OV infection. We extended these studies to test the hypothesis that epigenetic modulators could alter the antiviral state of neuroblastoma cells to enhance OV killing. Pre-treatment with the DNA methyltransferase inhibitor 5-Azacytidine enhanced P/V virus-mediated death of SK-N-AS cells in a dose- and MOI-dependent manner. While killing of SK-N-AS cells by the P/V virus alone was minimally caspase-dependent, combined treatment with 5-Azacytidine and P/V virus shifted cell killing to a largely caspase-dependent mechanism. 5-Azacytidine pre-treatment of SK-N-AS cells dampened P/V virus growth, which correlated with higher expression of antiviral mediators such as interferon-beta and OAS2. These studies demonstrate that antiviral responses mediated by a host factor (CD24) and by epigenetic modulators (5-Azacytidine) can be exploited to enhance tumor cell killing by RNA OVs.