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Incretins are gastric hormones released by intestinal K-cells in response to food consumption and stimulate insulin secretion from pancreatic beta cells. One of these hormones, glucose-dependent insulinotropic peptide (GIP) is also anabolic in bone. Individuals with diabetes experience diminished bone quality caused by a low bone formation osteopenia. The present study seeks to identify a mechanism for diabetic osteopenia in which diabetes interferes with GIP-stimulated increases in the collagen cross-linking enzyme lysyl oxidase (LOX) in osteoblasts, leading to decreased collagen integrity and the trabecular abnormalities seen in diabetic bone. Micro-CT analysis and picrosirius red histology of long bones from LOX +/- and wild type mice made diabetic by low dose streptozotocin induction revealed a profound exacerbation of the decreased bone volume, impaired trabecular structure, and disorganized collagen matrix seen in diabetic mice when the mice were also haploinsufficient for LOX. Furthermore, qPCR of RNA isolated from diabetic long bones revealed a more than 20 fold decrease in LOX expression in diabetic bone from wild type mice. Treatment of wild type osteoblasts in culture with GIP results in a significant increase in LOX transcript and protein levels. Interestingly in our diabetic mice there is a decrease in osteoblast derived LOX and an abnormal increase in serum levels of the anti-incretin gut-derived dopamine, which is known to inhibit the effects of GIP in the pancreas. Therefore the ability of dopamine to inhibit GIP-stimulated signaling in osteoblasts was examined. Data indicate a strong dose-dependent inhibition of GIP-stimulated LOX expression when primary osteoblast cultures are pretreated with dopamine. Finally, pretreatment of primary osteoblasts with the dopamine receptor inhibitor amisulpride restored the impaired GIP stimulated increases in LOX expression in osteoblasts isolated from diabetic mice. This study defines a potential mechanism for diabetic bone disease and suggests that interference with dopamine signaling would likely restore bone health in diabetes.

INTRODUCTION: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study was to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. METHODS: In vitro: The effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays, gene knock down and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effects of PXS-S1C on cancer cell expression of LOXL2 and proliferation were determined. To find potential LOXL2 substrates, carbonyl-containing proteins of gingival fibroblasts treated with CM +/- PXS-S1C were affinity-labeled and then purified by affinity chromatography and identified by western blot. In vivo: The effects of PXS-S1C on cancer cell growth and metastasis in vivo were investigated using orthotopic oral tongue cancer mouse models in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumor growth was monitored by both caliper measurement and in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemical staining with proliferation markers. RESULTS: PXS-S1C significantly inhibited gingival fibroblast proliferation stimulated by tumor cell CM and attenuated phosphorylation of PDGFRβ at the Y771 and Y857, but not Y751 residues in response to CM treatment. PXS-S1C inhibited ERK1/2-signaling in fibroblasts but not AKT pathway in response to CM treatment. PDGFR activation by oral tumor cells was mimicked by PDGF-AB but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 in HSC3 oral cancer cells in vitro, suggesting the existence of a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified PDGFR. In vivo studies: Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 by injections of PXS-S1C significantly decreased both progression and metastasis of oral cancers in vivo in both mouse models. Mice without PXS-S1C treatment developed larger tongue volumes (p

Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90% of these patients, yet the mechanisms underlying this event remain poorly defined. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to suppress tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-beta from a tumor suppressor to a tumor promoter through molecular mechanisms that remain incompletely understood. Changes in tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the activity of the extracellular crosslinking enzyme, lysyl oxidase (LOX), which regulates desmoplastic reactions and metastasis. We show that TGF-beta induces the synthesis and secretion of LOX from normal and malignant MECs, and in breast cancers produced in mice. Additionally, antagonizing LOX activity reduces TGF-beta-mediated invasion and epithelial-mesenchymal transition (EMT) in breast cancer cells. We further show that increased extracellular matrix (ECM) rigidity promotes the proliferation of malignant MECs, a cellular reaction that is abrogated by inhibiting TGF-beta signaling or by antagonizing LOX activity. As a possible mechanism for the influence of matrix rigidity on TGF-beta signaling, we sought to define the microRNA expression profiles induced by TGF-beta and ECM rigidity during metastatic progression. Through global profiling analyses, we identified microRNAs whose expression was induced by TGF-beta and associated with metastatic potential. In doing so, we identified miR-181a as a TGF-beta-regulated “metastamir” that enhanced the metastatic potential of breast cancers by promoting their acquisition of EMT, migratory, and invasive phenotypes. Mechanistically, inactivating miR-181a elevated the expression of the pro-apoptotic molecule, Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a activity was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression is dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and is highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings implicate LOX and miR-181a expression as novel diagnostic markers for metastatic progression, as well as an innovative therapeutic targets to treat metastatic breast cancers.

Several members of the lysyl oxidase family of copper-dependent amine oxidases have been implicated in tumor development. The Iysyl oxidase (LOX) and LOX-like 2 (LOXL2) genes have been mapped to chromosomal regions affected by loss of heterozygosity (LOH) in several cancers, including those of the colon and esophagus. Indeed, there have been numerous reports of reduced LOX and a few reports of reduced LOXL2 expression in various cancers. Identification of microsatellite markers within the LOX locus and the LOXL2 gene allowed for evaluation ofthe status of these gene alleles in colon and esophageal tumors. There was significant LOH of the LOX locus in colon tumors that was accompanied by reduced mRNA expression and a spectrum of alterations and mutations affecting the LOX gene. This study demonstrated, for the first time, that genetic events, namely LOH, deletions and mutations ofthe LOX gene, were responsible, at least partly, for the reduction of LOX gene expression. There was also significant LOH of the LOXL2 gene in both colon and esophageal tumors. However, instead of a reduction of LOXL2 expression, there was increased expression that correlated with less differentiated tumors and absent elastosis, both indicators of poor prognosis. Further studies indicated that both LOX and LOXL2 are absent in non-invasive tumor cell lines but re-expressed in invasive cell lines, likely as part of the thelial-mesenchymal transition that occurs in the last steps of tumorigenesis to facilitate metastasis. The results presented and research strategy outlined in this dissertation will define the importance of LOXL2 amine oxidase activity and protein interactions in the critical but poorly understood process oftumor cell migration and invasion.