Metastasis and recurrent tumors caused by drug resistance are the main reasons for breast-cancer associated deaths. The mechanisms are still not fully understood. In this study, we address novel mechanisms underlying which localized cancer metastasis turns wildly aggressive to multiple organs during anticancer treatment. We show that Krüppel-like factor 8 (KLF8) expression can be over-induced by chemotherapeutic drugs. Data from large-cohorts of patients shows that after chemotherapy there is a close correlation between the aberrant high levels of KLF8 and C-X-C chemokine receptor type 4 (CXCR4) with drug resistance, metastasis, and poor prognosis. To mimic the drug-induced upregulation, KLF8 or CXCR4 was overexpressed in the lung-specific metastatic cell line LM2. We unexpectedly found that KLF8 or CXCR4 overexpressing cells metastasized extensively to multiple organs, particularly the C-X-C motif chemokine ligand 12 (CXCL12)-rich organs. We found that KLF8 or CXCR4 overexpression enhanced angiogenesis involving increased expression and secretion of vascular endothelial growth factor (VEGF) protein. We also found that KLF8 or CXCR4 overexpression strongly enhanced the formation of filopodia-like protrusions (FLPs) and proliferation. In addition, we opened a new avenue for developing an effective anticancer therapy against Herceptin-resistance, which is a major problem during the treatment of human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. Hypoxia-inducible factor 1 subunit alpha (HIF-1α) plays a critical role in drug-resistance. However, no active drug that can selectively target HIF-1α is available. Cerium oxide nanoparticles (CNPs) have recently emerged as anticancer agents with minimal side effects on normal tissues. We proved that CNPs showed a strong ability to sensitize Herceptin-resistant breast cancer cells to the cytotoxicity of Herceptin. Herceptin combined with CNPs was shown to effectively eliminate Herceptin-resistant breast cancer cells via HIF-1α and VEGF signaling suppression. We suggest great potential for their combination to overcome drug resistance, metastasis, and tumor relapse.

Cancer remains one of the most common causes of mortality around the world and continues to evade standardization of diagnostic and therapeutic guidelines. As a multifaceted and heterogenous disease, development of standard screening options and monitoring cancer progression in patients is a challenge. In this study, we hypothesized that this challenge could be addressed by a biomarker that plays an essential and central role in oncogenesis across multiple cancer types. Previous studies from our labs and others have shown that Chaperonin Containing TCP-1 (CCT) is present in multiple cancers and plays an important role in oncogenesis as a protein folding complex of many oncogenes. Increased CCT levels in tissues correlate with increased cancer stage and severity, while inversely correlating with patient outcomes. For this project, we investigated the potential of the second subunit, CCT2, as a biomarker for adult cancers and pediatric cancers including breast cancer, lung cancer, and neuroblastoma. While most cancers have high levels of CCT, we used bioinformatics and histological studies to build a foundation for which cancers would benefit the most from our studies and were available for testing the feasibility of CCT2 as a biomarker. Since CCT2 has been studied in adult cancers previously, we first used breast cancer cells spiked in blood to adjust CCT2 as a biomarker for liquid biopsy. Once we determined a range of use for CCT2 in liquid biopsy, we tested it in lung cancer patients. Both showed increased recovery and promising circulating cells that were cytokeratin negative and CCT2 positive. To determine if CCT2 would also work in cancers with very different origins, we looked at pediatric cancers. We used neuroblastoma as our test model to determine if the CCT complex had a similar significance and role in pediatric cancers as in adult cancers. We completed overexpression and knockdown studies which showed results that were congruent with what CCT's role in adult cancer has been. We therefore tested sensitivity to CCT drug inhibition and sensitivity in liquid biopsy detection and determined that CCT is vital for tumorigenesis in neuroblastoma. These results show that CCT has the unique potential as a novel biomarker for cancer detection and that the second subunit could be used as a readout for the presence of the entire complex.

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of < 8%. Risk factors of pancreatic cancer, such as chronic pancreatitis, help to elicit a pro-tumor immune response, and highly fibrotic environment that promotes tumorigenesis. To study how chronic pancreatitis promotes cancer initiation, traditional KRasG12D mice and double mutant Akt1Myr/KrasG12D mice were used to model microenvironment changes. Akt1Myr/KrasG12D mice were more susceptible to chronic tissue damage, accelerated tumor development and metastatic disease. These mice exhibited histological changes consistent with immune cell privilege, where M2 macrophages and non-cytotoxic eosinophils were co-localized with fibrotic regions. IL-5 expression was up regulated in pancreatic cells undergoing acinar to ductal metaplasia and then diminished in advanced lesions. Tumor cells treated with IL-5 exhibit increased migration and activation through STAT5 signaling. Collectively, the results suggest that eosinophils, which are responsive to IL-5, are key mediators in the pancreatic environment subjected to chronic inflammation and injury. Current therapeutics fall short in increasing patient survival. There remains an urgent need for innovative treatments and thus we tested difluoromethylornithine (DFMO) in combination with a novel polyamine transport inhibitor, Trimer44NMe, against Gemcitabine-resistant PDAC cells. Prior clinical failures when targeting polyamine biosynthesis with DFMO monotherapy may be due to tumor escape via an undefined polyamine transport system. In pancreatic tumor cells DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or cell cycle arrest. In vivo orthotopic PDAC growth with DFMO treatment resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice, and increased M1 macrophage infiltration and reduced FoxP3 expression. Collectively, these studies demonstrate that targeting polyamine pathways in PDAC is a promising immunomodulating therapy that increases survival.

Colorectal cancer (CRC) is the third most common and third deadliest cancer worldwide with rising incidence rates attributed to environmental risk factors like diet. Investigating how these factors impact carcinogenesis requires an understanding of how transcriptional events evolve with respect to neoplastic progression. We employed meta-transcriptomics and latent trajectory modeling to establish a compendium of profiled healthy, adenoma, and CRC samples scored by their position along a pseudotemporal axis. By interpolating a continuous scale from cross-sectional data, dynamic processes occurring throughout disease progression can be analyzed more accurately. For example, smaller pseudotimes represented pre-malignant dysplasia and was characterized by cellular hyperproliferation downstream of genomic stress. Larger pseudotimes represented post-malignant progression and was characterized by a prominent stromal and inflammatory response. As dysregulated bile acid (BA) metabolism is intrinsically linked with diet and CRC development, we next assessed how neoplastic progression modulated colonic BA-related pathways. Pseudotemporal analysis delineated a role and clinical utility of the FGFR4-KLB-FGF19 pathway in disease progression. FGFR4 was an early CRC oncogene and the only FGFR that could be directly associated with tumorigenesis, suggesting that targeted inhibition may be a novel therapeutic modality. KLB's expression and prognostic profile was inverse to FGFR4, indicating a tumor suppressor role in early CRC. In particular, predictive informatics ascribed a functional role for KLB in opposing FGFR4-mediated dysplastic processes, which was validated using cellular models of differentiation as well as transgenic and morphological studies. FGF19 was also identified as an oncogene and putative blood-based CRC biomarker due to its endocrine properties. Immunodeficient mice transplanted with FGF19-expressing cells demonstrated supraphysiologic levels of circulating FGF19 that exerted potent endocrine effects targeting hepatic metabolism and enterohepatic recirculation of BAs. Collectively, the data provide clear evidence for the importance of the FGFR4-KLB-FGF19 complex in modulating CRC oncogenesis as well as its potential translational applicability for screening/diagnostic purposes.

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abstract: Cancer is a serious health concern. Current treatments are limited due to certain subpopulations of cancer cells being resistant to chemotherapy and radiation. These subpopulations have been qualitatively identified but much work remains to quantify the abnormalities they exhibit such as irregular nuclear shape. This dissertation seeks to determine physical science methods which can identify and quantify the biological characteristics of cancer and non-cancer cells. For the first project, the deoxyribonucleic acid (DNA) and chromatin of cancer and non-cancer esophageal cells were quantified using spectrophotometry and atomic force microscopy. Then the cellular nucleus shape, chromocenters, nucleoli, and nuclear speckles were characterized using 3-D confocal microscopy. A majority of a cell's DNA is isolated in the supernatant fraction during salt fractionation for both cancer and non-cancer. Additionally, the nuclear size of cancer cells is roughly twice that of non-cancer cells due to the increased ploidy of the cancer cell line (more chromatin) and this chromatin exists in a less decondensed state than that of the chromatin in non-cancer cells. Then using combined atomic force microscopy and CLSM, the Young's modulus of cancer stem-like cells and non-stem-like cells were characterized for three breast cell lines: MDA-MB-231, MCF-7, and MCF-10A. It was determined that the MCF-7 is impacted by buffer environment whereas the MDA-MB-231 and the MCF-10A cell lines are not. MCF-7 cells are stiffer when measured in Phosphate Buffer Solution (PBS) compared to Hank's Balanced Salt Solution (HBSS) buffer possibly due to the fact that HBSS buffer tends to enhance the Warburg effect on cell lines. Additionally, there is a significant stiffness difference between stem cells and non-stem cells in the MCF-7 cell line which does not occur in the MDA-MB-231 cell line for the larger tip. These differences could be attributed to differences in cell phenotype for the cell lines. MDA-MB-231 cells are mesenchymal so it agrees with the hypothesis that there is no difference between cancer stem cells (CSCs) and non-CSCs cell stiffness; on the other hand the MCF-7 cell line is luminal so the CSCs being more mesenchymal-like would be softer than the non-CSCs. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

Anti-inflammatory specific cytokines and chemokines are elevated in many advanced tumors and correlate with poor prognosis. However, the differential expression of anti-inflammatory cytokines and chemokines in prostate cancer is not known. We investigated the hypotheses that androgen unresponsive DU145 and PC3 prostate cancer cells and androgen responsive LNCaP prostate cancer cells, differentially expressed selected anti-inflammatory and pro-inflammatory cytokines and chemokines and that, dendritic cells pulsed with prostate tumor antigens will induce mainly pro-inflammatory cytokines and chemokines in T cells using mouse models. Our results indicated that anti-inflammatory specific cytokines IL-1 0, IL-4, and anti-inflammatory specific chemokine CCL- 17 (TARC) and cognate receptor CCR4 are expressed in prostate cancer cell lines. Quantitative real-time PCR (qRT-PCR) revealed an almost five-fold increase in chemokine CCL17 and its cognate receptor CCR4 mRNA in androgen unresponsive DU145 and PC3 prostate cancer cell lines compared to androgen responsive prostate tumor LNCaP. Protein analysis indicated significantly increased secretion of anti-inflammatory cytokine IL- 10 by DU145 and PC3 compared to LNCaP. Furthermore, pro-inflammatory cytokine IFN-y and pro-inflammatory chemokine IP- 10 secretion were significantly less in these prostate cancer cells, when compared to immortalized normal prostate epithelial cells. Our in- vivo analysis revealed that T cells were activated by pulsed dendritic cells shown in the increase mRNA expression of pro-inflammatory cytokine IFN-y and pro-inflammatory chemokine IP- 10, and cognate receptor CXCR3. However, a predominant pro-inflammatory response was not observed as anti-inflammatory cytokines and chemokines were also seen. The production of anti-inflammatory cytokines and chemokines suggests a possible mechanism for prostate cancer to evade host immune responses by negatively modulating immune responses that are necessary for destroying cancers cells.. Cytokine and chemokine profiles could be used as potential prognostic markers for disease progression. Additionally, an effacious vaccine will depend on its ability to inhibit the recruitment of known distinct functional anti-inflammatory effector molecules, implicated in prostate cancer progression.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors, characterized by a highly invasive cell population. GB tumors develop treatment resistance and ultimately recur; the median survival is nearly fifteen months and importantly, the invading cell population is attributed with having a decreased sensitivity to therapeutics. Thus, there remains a necessity to identify the genetic and signaling mechanisms that promote tumor spread and therapeutic resistance in order to develop new targeted treatment strategies to combat this rapidly progressive disease. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival, and is dependent upon the activity of multiple Rho GTPases including Rac1. Here, we show that Cdc42 is essential in Fn14-mediated Rac1 activation. We identified two guanine nucleotide exchange factors (GEFs), Ect2 and Trio, involved in the TWEAK-induced activation of Cdc42 and Rac1, respectively, as well as in the subsequent TWEAK-Fn14 directed glioma cell migration and invasion. In addition, we characterized the role of SGEF in promoting Fn14-induced Rac1 activation. SGEF, a RhoG-specific GEF, is overexpressed in GB tumors and promotes TWEAK- Fn14-mediated glioma invasion. Moreover, we characterized the correlation between SGEF expression and TMZ resistance, and defined a role for SGEF in promoting the survival of glioma cells. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-B dependent manner and inhibition of SGEF expression sensitizes glioma cells to TMZ treatment. Lastly, gene expression analysis of SGEF depleted GB cells revealed altered expression of a network of DNA repair and survival genes. Thus TWEAK-Fn14 signaling through the GEF-Rho GTPase systems which include the Ect2, Trio, and SGEF activation of Cdc42 and/or Rac1 presents a pathway of attractive drug targets in glioma therapy, and SGEF signaling represents a novel target in the setting of TMZ refractory, invasive GB cells.

This thesis describes the characterisation of human MRCKcc (myotonic dystrophy kinase-related Cdc42-binding kinase) and the signalling role of MRCK in tumour cell invasion. The first results chapter reports the identity and sequence of the human isoform of MRCKa. It is shown that previously reported expression of a sequence known as PK428 in breast tumours with poor prognosis represents MRCKa expression. PK428 is identified as a partial cDNA of the previously uncloned human MRCKa. The cloning of several splice-variants of full-length MRCKa is described. MRCKa expression is also analysed in human tumour samples, but the data are not conclusive enough to establish MRCKa expression as a prognostic marker. Subsequent work details the functional role of MRCKa and MRCK? in mediating invasion of three-dimensional extracellular matrices by tumour cell lines. In tumour cells that exhibit an elongated morphology, abrogation of signalling of both MRCKa/?, by RNA interference, and ROCK (Rhokinase), with the small-molecule inhibitors Y-27632 or HA-1077, results in inhibition of invasion. This is associated with a collapsed cellular morphology and abrogation of phosphorylation of both the myosin targeting subunit (MYPT1) of myosin light chain phosphatase and of myosin light chain (MLC2). Effects on morphology and invasion are recapitulated with blebbistatin, a specific inhibitor of actomyosin contractility. This suggests that MRCK and ROCK have a redundant role in promoting invasion through regulation of myosin activity. In a cell line with a rounded morphology in threedimensional matrices, switching between three morphological states is identified - rounded, elongated and collapsed, in order of decreasing actomyosin contractility. In this cell line, MRCK and ROCK have a redundant role in maintaining the elongated morphology but ROCK alone signals the high level of contractility that drives the rounded morphology.