The Effect of a Histone Deacetylase Inhibitor (AR-42) and Zoledronic Acid on Adult T-Cell Leukemia/Lymphoma Osteolytic Bone Tumors.

Adult T-cell leukemia/lymphoma (ATL) is an intractable disease affecting nearly 4% of Human T-cell Leukemia Virus Type 1 (HTLV-1) carriers. Acute ATL has a unique interaction with bone characterized by aggressive bone invasion, osteolytic metastasis, and hypercalcemia. We hypothesized that dual tumor and bone-targeted therapies would decrease tumor burden in bone, the incidence of metastasis, and ATL-associated osteolysis. Our goal was to evaluate dual targeting of both ATL bone tumors and the bone microenvironment using an anti-tumor HDACi (AR-42) and an osteoclast inhibitor (zoledronic acid, Zol), alone and in combination. Our results showed that AR-42, Zol, and AR-42/Zol significantly decreased the viability of multiple ATL cancer cell lines in vitro. Zol and AR-42/Zol decreased tumor growth in vivo. Zol ± AR-42 significantly decreased ATL-associated bone resorption and promoted new bone formation. AR-42-treated ATL cells had increased mRNA levels of PTHrP, ENPP2 (autotaxin) and MIP-1α, and TAX viral gene expression. AR-42 alone had no significant effect on tumor growth or osteolysis in mice. These findings indicate that Zol adjuvant therapy has the potential to reduce growth of ATL in bone and its associated osteolysis.

Effect of Dickkopf-1 (Dkk-1) and SP600125, a JNK Inhibitor, on Wnt Signaling in Canine Prostate Cancer Growth and Bone Metastases.

Human Dickkopf-1 (Dkk-1) upregulates a noncanonical Wnt/JNK pathway, resulting in osteoclast stimulation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) of cancer cells. Ace-1-Dkk-1, a canine prostate cancer (PCa) cell line overexpressing Dkk-1, was used to investigate Wnt signaling pathways in PCa tumor growth. SP600125, a JNK inhibitor, was used to examine whether it would decrease tumor growth and bone tumor phenotype in canine PCa cells in vitro and in vivo. Ace-1-VectorYFP-Luc and Ace-1-Dkk-1YFP-Luc cells were transplanted subcutaneously, while Ace-1-Dkk-1YFP-Luc was transplanted intratibially into nude mice. The effects of Dkk-1 and SP600125 on cell proliferation, in vivo tumor growth, and bone tumor phenotype were investigated. The mRNA expression levels of Wnt/JNK-related genes were measured using RT-qPCR. Dkk-1 significantly increased the mRNA expression of Wnt/JNK-signaling-related genes. SP600125 significantly upregulated the mRNA expression of osteoblast differentiation genes and downregulated osteoclastic-bone-lysis-related genes in vitro. SP600125 significantly decreased tumor volume and induced spindle-shaped tumor cells in vivo. Mice bearing intratibial tumors had increased radiographic density of the intramedullary new bone, large foci of osteolysis, and increased cortical lysis with abundant periosteal new bone formation. Finally, SP600125 has the potential to serve as an alternative adjuvant therapy in some early-stage PCa patients, especially those with high Dkk-1 expression.

Parathyroid hormone-related protein promotes bone loss in T-cell leukemia as well as in solid tumors.

Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) are important factors that increase bone resorption and hypercalcemia in adult T-cell leukemia (ATL). We investigated the role of PTHrP and MIP-1α in the development of local osteolytic lesions in T-cell leukemia through overexpression in Jurkat T-cells. Injections of Jurkat-PTHrP and Jurkat-MIP-1α into the tibia and the left ventricle of NSG mice were performed to evaluate tumor growth and metastasis in vivo. Jurkat-pcDNA tibial neoplasms grew at a significantly greater rate and total tibial tumor burden was significantly greater than Jurkat-PTHrP neoplasms. Despite the lower tibial tumor burden, Jurkat-PTHrP bone neoplasms had significantly greater osteolysis than Jurkat-pcDNA and Jurkat-MIP-1α neoplasms. Jurkat-PTHrP and Jurkat-pcDNA cells preferentially metastasized to bone following intracardiac injection, though the overall metastatic burden was lower in Jurkat-PTHrP mice. These findings demonstrate that PTHrP induced pathologic osteolysis in T-cell leukemia but did not increase the incidence of skeletal metastasis.

Mouse model recapitulates the phenotypic heterogeneity of human adult T-cell leukemia/lymphoma in bone.

Adult T-cell leukemia/lymphoma has a unique relationship to bone including latency in the marrow, and development of bone invasion, osteolytic tumors and humoral hypercalcemia of malignancy. To study these conditions, we established and characterized a novel mouse model of ATL bone metastasis. Patient-derived ATL cell lines including three that do not express HTLV-1 oncoprotein Tax (ATL-ED, RV-ATL, TL-Om1), an in vitro transformed human T-cell line with high Tax expression (HT-1RV), and an HTLV-1 negative T-cell lymphoma (Jurkat) were injected intratibially into NSG mice, and were capable of proliferating and modifying the bone microenvironment. Radiography, µCT, histopathology, immunohistochemistry, plasma calcium concentrations, and qRT-PCR for several tumor-bone signaling mRNAs were performed. Luciferase-positive ATL-ED bone tumors allowed for in vivo imaging and visualization of bone tumor growth and metastasis over time. ATL-ED and HT-1RV cells caused mixed osteolytic/osteoblastic bone tumors, TL-Om1 cells exhibited minimal bone involvement and aggressive local invasion into the adjacent soft tissues, Jurkat cells proliferated within bone marrow and induced minimal bone cell response, and RV-ATL cells caused marked osteolysis. This mouse model revealed important mechanisms of human ATL bone neoplasms and will be useful to investigate biological interactions, potential therapeutic targets, and new bone-targeted agents for the prevention of ATL metastases to bone.

Animal Models of Cancer-Associated Hypercalcemia.

Cancer-associated hypercalcemia (CAH) is a frequently-occurring paraneoplastic syndrome that contributes to substantial patient morbidity and occurs in both humans and animals. Patients with CAH are often characterized by markedly elevated serum calcium concentrations that result in a range of clinical symptoms involving the nervous, gastrointestinal and urinary systems. CAH is caused by two principle mechanisms; humorally-mediated and/or through local osteolytic bone metastasis resulting in excessive calcium release from resorbed bone. Humoral hypercalcemia of malignancy (HHM) is the most common mechanism and is due to the production and release of tumor-associated cytokines and humoral factors, such as parathyroid hormone-related protein (PTHrP), that act at distant sites to increase serum calcium concentrations. Local osteolytic hypercalcemia (LOH) occurs when primary or metastatic bone tumors act locally by releasing factors that stimulate osteoclast activity and bone resorption. LOH is a less frequent cause of CAH and in some cases can induce hypercalcemia in concert with HHM. Rarely, ectopic production of parathyroid hormone has been described. PTHrP-mediated hypercalcemia is the most common mechanism of CAH in human and canine malignancies and is recognized in other domestic species. Spontaneous and experimentally-induced animal models have been developed to study the mechanisms of CAH. These models have been essential for the evaluation of novel approaches and adjuvant therapies to manage CAH. This review will highlight the comparative aspects of CAH in humans and animals with a discussion of the available animal models used to study the pathogenesis of this important clinical syndrome.

HTLV-1 viral oncogene HBZ induces osteolytic bone disease in transgenic mice.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

The Effect of a Histone Deacetylase Inhibitor (AR-42) on Canine Prostate Cancer Growth and Metastasis.

BACKGROUND: Canine prostate cancer (PCa) is an excellent preclinical model for human PCa. AR-42 is a histone deacetylase inhibitor (HDACi) developed at The Ohio State University that inhibits the proliferation of several cancers, including multiple myeloma, lung, and hepatocellular cancer. In this study, we investigated whether AR-42 would prevent or decrease. The growth and metastasis of a canine PCa (Ace-1 cells) to bone in vitro and in vivo. METHODS: Proliferation, cell viability, invasion, and metastasis of a canine prostate cancer cell line (Ace-1) were measured following treatment with AR-42. Expression of anoikis resistance, epithelial-to-mesenchymal transition (EMT), and stem cell-related markers were also evaluated. To assess the efficacy of AR-42 on prevention of PCa metastasis to bone, Ace-1 cells were injected in the left cardiac ventricle of nude mice, mice were treated with AR-42, and the incidence and growth of bone metastasis were measured. Bioluminescence was performed to monitor the bone metastases in nude mice. RESULTS: AR-42 inhibited the in vitro proliferation of Ace-1 cells in a time- and dose-dependent manner. The IC50 concentration of AR-42 for Ace-1 cells was 0.42 µM after 24 hr of treatment. AR-42 induced apoptosis, decreased cell migration, and increased the stem cell properties of Ace-1 cells in vitro. AR-42 downregulated E-cadherin, N-cadherin, TWIST, MYOF, anoikis resistance, and osteomimicry genes, while it upregulated SNAIL, PTEN, FAK, and ZEB1 gene expression in Ace-1 cells. Importantly, AR-42 decreased the bioluminescence and incidence of bone metastasis in nude mice. In addition, AR-42 induced apoptosis and altered the tumor cell morphology to an irregular cell phenotype with condensed chromatin in the bone metastases. CONCLUSION: AR-42 decreased PCa growth and bone metastasis, induced apoptosis, and downregulated osteomimicry genes in PCa cells in the bone microenvironment. Prostate 77:776-793, 2017. © 2017 Wiley Periodicals, Inc.

Gamma(2)-melanocyte stimulating hormone decreases food intake in chicks.

The role of gamma melanocyte stimulating hormone (gamma-MSH) in appetite regulation is controversial in mammals and to our knowledge unreported within the avian class. Thus, the present study was designed to determine the effects of intracerebroventricularly (i.c.v.) administered gamma2-MSH on food intake using Cobb-500 chicks as models. In Experiment 1, chicks that received i.c.v. gamma2-MSH decreased their food intake throughout the 180 min observation period and plasma glucose concentration was not affected. Water intake was also decreased in i.c.v. gamma2-MSH-treated chicks, but only from 30 to 90 min post-injection. In Experiment 2, food pecking efficiency was decreased in i.c.v. gamma2-MSH-treated chicks and the amount of time spent sitting was increased. Other behaviors were not significantly affected by i.c.v. gamma2-MSH including distance traveled, the number of jumps, escape attempts, defecations, food pecks, exploratory pecks, and the amount of time spent standing, preening, perching, or in deep rest. These data suggest that gamma2-MSH is associated with anorexigenic effects and because of gamma-MSH's selectivity, implicates the melanocortin 3 receptor in appetite regulation.