The Wnt/β-catenin signaling pathway is often deregulated in cancer. Here we investigate Wnt/β-catenin signaling, aberrant accumulation of β-catenin, and epigenetic deregulation in breast cancer and parathyroid tumours. An aberrantly spliced Wnt coreceptor LRP5 (LRP5Δ) is important for accumulation of nonphosphorylated active β-catenin and tumour growth in parathyroid tumours. Paper I demonstrated frequent expression of LRP5Δ in breast tumours and substantiated that breast tumour cell growth was dependent on continuous activation of the Wnt/β-catenin pathway by LRP5Δ. A LRP5 antibody reduced the levels of active β-catenin, inhibited tumour cell growth and caused apoptosis in breast cancer cells. Antibody therapy may have a significant role in the treatment of breast cancer. Paper II revealed lost expression of the tumour suppressor gene APC in parathyroid carcinomas, likely due to CpG methylation. Also accumulation of nonphosporylated active β-catenin was observed, indicating activation of Wnt/β-catenin signaling. Treatment of primary parathyroid carcinoma cells with the demethylating agent 5-aza-2’-deoxycytidine reduced the levels of active β-catenin, inhibited cell growth and caused apoptosis, suggesting that adjuvant epigenetic therapy could be considered in patients with metastatic or recurrent parathyroid carcinoma. In paper III we showed that the expression of the tumour suppressor gene HIC1 was generally reduced in parathyroid tumours of primary and secondary origin, and parathyroid carcinomas. Overexpressing HIC1 reduced cell viability and suppressed colony formation, supporting a tumour suppressor role in the parathyroid gland. Results suggested that the observed underexpression of HIC1 could be explained by epigenetic deregulation involving histone methylation rather than CpG methylation. Paper IV demonstrated increased expression of the histone methyltransferase EZH2 in parathyroid tumours of primary and secondary origin, and most apparent in parathyroid carcinomas. Decreasing EZH2 resulted in reduced cell viability and colony formation capacity suggesting that EZH2 may function as an oncogene in parathyroid tumours. Furthermore, depletion of EZH2 also reduced the amount of active β-catenin. EZH2 may represent a novel therapeutic target in parathyroid tumours. The fact that HIC1 was underexpressed and EZH2 overexpressed in parathyroid tumours regardless of the hyperparathyroid disease state may represent a possibility for a common pathway in parathyroid tumour development.

Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity. To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.

Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity. To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.

Canonical Wnt signaling is involved in many aspects of development including axis specification and anterior-posterior neuroectoderm formation during vertebrate embryogenesis. Axin2, a homologue of Axin1, is thought to have a similar regulatory role within the cell, but differences in their expression and binding partners suggest Axin2 is not completely redundant with Axin1. To better understand Axin2 in canonical Wnt signaling, I utilized several approaches to explore its expression and function. In the zebrafish embryo, I found Axin2 is expressed in known active domains of Wnt signaling, suggesting an inducible regulatory role. Additionally, canonical Wnt signaling was sufficient and necessary to induce Axin2 expression and Axin2 was sufficient and necessary to inhibit Wnt signaling. As Wnt signaling is important in development and its dysregulation has been implicated in diseases such as colorectal cancer, this study helps advance our understanding of how Wnt signaling regulates itself through the use of negative feedback inhibitors, such as Axin2.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a common location for breast cancer cell metastasis, and progression of tumor in bone can lead to bone loss and affect human health. Osteocytes have important roles in bone homeostasis and osteogenesis, and their interaction with metastasized cancer cells are known to affect progression of metastasized tumor. However, the potential role of metabolic signaling and actin- cytoskeleton-associated moesin in the interaction of osteocytes and tumor cells remain poorly understood. In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration. Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.

Considering that both cardiovascular disease (CVD) and congenital heart diseases (CHD) are still the leading cause of morbidity and mortality worldwide, there is a need for a robust and reliable cardiac model. Cardiac organoids are complex, three-dimensional cellular constructs that recapitulate the processes of the human embryonic heart. However, certain vital morphological features within the fetus are not yet replicable with cardiac organoids. Here we report our investigation to generate cardiac organoids with chamber formations. Our method involves modulating the Wnt pathway at two different instances while also implementing two cell seeding densities, all to determine the most optimized that to produce chamber formations within cardiac organoids.

The nematode, Caenorhabditis elegans is an ideal animal model to study conserved mechanisms of developmental and postdevelopmental processes. Here, I describe the role of an Axin family member, pry-1, in aging, lipid metabolism, and seam cell development. Our analysis of pry-1 animals showed a catastrophic collapse of adult lifespan, which was accompanied with hallmarks of accelerated aging. Transcriptome profiling of pry-1 mutants revealed altered expression of genes associated with aging and lipid metabolism such as vitellogenins, fatty acid desaturases, lipases, fatty acid transporters and genes involved in cuticle synthesis. Consistent with this, pry-1 animals display significantly reduced levels of somatic lipids. Knockdowns of vitellogenins in the pry-1 background restored lifespan and lipid levels, suggesting that vitellogenins are necessary to mediate pry-1 function in aging and lipid metabolic processes. Additionally, lowered expression of desaturases and lipidomics analysis provided evidence of reduced fatty acid synthesis in pry-1 animals. In agreement with this, an exogenous supply of oleic acid restored depleted lipids in somatic tissues in addition to suppressing the short-lived phenotype of worms. In addition, transcriptome profiling for differentially expressed miRNAs in pry-1(mu38) identified heterochronic miRNAs (lin-4 and let-7 -family members) to act downstream of pry-1 /Axin. In C. elegans, these miRNAs are known to robustly regulate the stem-like, seam cell division. Loss of pry-1 function caused heterochronic defects such that the seam cells divide precociously to produce additional cells. The pry-1-miRNAs are involved in mediating silencing of the heterochronic gene, hbl-1, a C. elegans hunchback homolog, to regulate seam cell division. Furthermore, I report identification of novel miRNAs from C. elegans and C. briggsae. Overall, our findings demonstrate a novel role of the Wnt signaling regulator, pry-1/Axin, in the maintenance of adult lifespan that involves lipid homeostasis and regulation of heterochronic miRNA to control the developmental timing of seam cell division in C. elegans. / Thesis / Doctor of Philosophy (PhD)

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