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.

In Vivo Tumorigenesis, Osteolytic Sarcomas, and Tumorigenic Cell Lines from Transgenic Mice Expressing the Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Viral Oncogene.

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia, a disease commonly associated with hypercalcemia and osteolysis. There is no effective treatment for HTLV-1, and the osteolytic mechanisms are not fully understood. Mice expressing the HTLV-1 oncogene Tax, driven by the human granzyme B promoter (Tax+), develop osteolytic tumors. To investigate the progression of the bone-invasive malignancies, wild-type, Tax+, and Tax+/interferon-γ-/- mice were assessed using necropsy, histologic examination, IHC analysis, flow cytometry, and advanced imaging. Tax+ and Tax+/interferon-γ-/- malignancies of the ear, tail, and foot comprised poorly differentiated, round to spindle-shaped cells with prominent neutrophilic infiltrates. Tail tumors originated from muscle, nerve, and/or tendon sheaths, with frequent invasion into adjacent bone. F4/80+ and anti-mouse CD11b (Mac-1)+ histiocytic cells predominated within the tumors. Three Tax+/interferon-γ-/- cell lines were generated for in vivo allografts, in vitro gene expression and bone resorption assays. Two cell lines were of monocyte/macrophage origin, and tumors formed in vivo in all three. Differences in Pthrp, Il6, Il1a, Il1b, and Csf3 expression in vitro were correlated with differences in in vivo plasma calcium levels, tumor growth, metastasis, and neutrophilic inflammation. Tax+ mouse tumors were classified as bone-invasive histiocytic sarcomas. The cell lines are ideal for further examination of the role of HTLV-1 Tax in osteolytic tumor formation and the development of hypercalcemia and tumor-associated inflammation.

Canine prostatic cancer cell line (LuMa) with osteoblastic bone metastasis.

BACKGROUND: Osteoblastic bone metastasis represents the most common complication in men with prostate cancer (PCa). During progression and bone metastasis, PCa cells acquire properties similar to bone cells in a phenomenon called osteomimicry, which promotes their ability to metastasize, proliferate, and survive in the bone microenvironment. The mechanism of osteomimicry resulting in osteoblastic bone metastasis is unclear. METHODS: We developed and characterized a novel canine prostatic cancer cell line (LuMa) that will be useful to investigate the relationship between osteoblastic bone metastasis and osteomimicry in PCa. The LuMa cell line was established from a primary prostate carcinoma of a 13-year old mixed breed castrated male dog. Cell proliferation and gene expression of LuMa were measured and compared to three other canine prostatic cancer cell lines (Probasco, Ace-1, and Leo) in vitro. The effect of LuMa cells on calvaria and murine preosteoblastic (MC3T3-E1) cells was measured by quantitative reverse-transcription polymerase chain reaction and alkaline phosphatase assay. LuMa cells were transduced with luciferase for monitoring in vivo tumor growth and metastasis using different inoculation routes (subcutaneous, intratibial [IT], and intracardiac [IC]). Xenograft tumors and metastases were evaluated using radiography and histopathology. RESULTS: After left ventricular injection, LuMa cells metastasized to bone, brain, and adrenal glands. IT injections induced tumors with intramedullary new bone formation. LuMa cells had the highest messenger RNA levels of osteomimicry genes (RUNX2, RANKL, and Osteopontin [OPN]), CD44, E-cadherin, and MYOF compared to Ace-1, Probasco, and Leo cells. LuMa cells induced growth in calvaria defects and modulated gene expression in MC3T3-E1 cells. CONCLUSIONS: LuMa is a novel canine PCa cell line with osteomimicry and stemness properties. LuMa cells induced osteoblastic bone formation in vitro and in vivo. LuMa PCa cells will serve as an excellent model for studying the mechanisms of osteomimicry and osteoblastic bone and brain metastasis in prostate cancer.

Canine Thyroid Cancer: Molecular Characterization and Cell Line Growth in Nude Mice.

Thyroid cancer is the most common endocrine malignancy in dogs. Dogs and humans are similar in the spontaneous development of thyroid cancer and metastasis to lungs; however, thyroid cancer has a higher incidence of metastasis in dogs. This study developed a preclinical nude mouse model of canine thyroid cancer using a canine thyroid adenocarcinoma cell line (CTAC) and measured the expression of important invasion and metastasis genes in spontaneous canine thyroid carcinomas and CTAC cells. CTAC cells were examined by electron microscopy. Short tandem repeat analysis was performed for both the original neoplasm and CTAC cells. CTAC cells were transduced with luciferase and injected subcutaneously and into the tail vein. Tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Invasion and metastasis genes were characterized in 8 follicular thyroid carcinomas (FTCs), 4 C-cell thyroid carcinomas, 3 normal thyroids, and CTAC cells. CTAC cells grew well as xenografts in the subcutis, and they resembled the primary neoplasm. Metastasis to the kidney and lung occurred infrequently following subcutaneous and tail vein injection of CTAC cells. STR analysis confirmed that CTAC cells were derived from the original neoplasm and were of canine origin. Finally, 24 genes were differentially expressed in spontaneous canine thyroid carcinomas, CTAC, and normal thyroids. This study demonstrated the usefulness of a nude mouse model of experimental canine thyroid carcinoma and identified potential molecular targets of canine follicular and C-cell thyroid carcinoma.

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.

HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The exact mechanism(s) through which latency and disease progression are regulated are not fully understood. CCCTC-binding factor (CTCF) is an 11-zinc finger, sequence-specific, DNA-binding protein with thousands of binding sites throughout mammalian genomes. CTCF has been shown to play a role in organization of higher-order chromatin structure, gene expression, genomic imprinting, and serve as a barrier to epigenetic modification. A viral CTCF-binding site (vCTCF-BS) was previously identified within the overlapping p12 (sense) and Hbz (antisense) genes of the HTLV-1 genome. Thus, upon integration, HTLV-1 randomly inserts a vCTCF-BS into the host genome. vCTCF-BS studies to date have focused primarily on HTLV-1 chronically infected or tumor-derived cell lines. In these studies, HTLV-1 was shown to alter the structure and transcription of the surrounding host chromatin through the newly inserted vCTCF-BS. However, the effects of CTCF binding in the early stages of HTLV-1 infection remains unexplored. This study examines the effects of the vCTCF-BS on HTLV-1-induced in vitro immortalization and in vivo viral persistence in infected rabbits. RESULTS: HTLV-1 and HTLV-1∆CTCF LTR-transactivation, viral particle production, and immortalization capacity were comparable in vitro. The total lymphocyte count, proviral load, and Hbz gene expression were not significantly different between HTLV-1 and HTLV-1∆CTCF-infected rabbits throughout a 12 week study. However, HTLV-1∆CTCF-infected rabbits displayed a significantly decreased HTLV-1-specific antibody response compared to HTLV-1-infected rabbits. CONCLUSIONS: Mutation of the HTLV-1 vCTCF-BS does not significantly alter T-lymphocyte transformation capacity or early in vivo virus persistence, but results in a decreased HTLV-1-specific antibody response during early infection in rabbits. Ultimately, understanding epigenetic regulation of HTLV-1 gene expression and pathogenesis could provide meaningful insights into mechanisms of immune evasion and novel therapeutic targets.

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.

HTLV-1 Tax-1 interacts with SNX27 to regulate cellular localization of the HTLV-1 receptor molecule, GLUT1.

An estimated 10-20 million people worldwide are infected with human T cell leukemia virus type 1 (HTLV-1), with endemic areas of infection in Japan, Australia, the Caribbean, and Africa. HTLV-1 is the causative agent of adult T cell leukemia (ATL) and HTLV-1 associated myopathy/tropic spastic paraparesis (HAM/TSP). HTLV-1 expresses several regulatory and accessory genes that function at different stages of the virus life cycle. The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products, and has also been demonstrated to play a key role in the pathogenesis of the virus. Several studies have identified a PDZ binding motif (PBM) at the carboxyl terminus of Tax-1 and demonstrated the importance of this domain for HTLV-1 induced cellular transformation. Using a mass spectrometry-based proteomics approach we identified sorting nexin 27 (SNX27) as a novel interacting partner of Tax-1. Further, we demonstrated that their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domains. SNX27 has been shown to promote the plasma membrane localization of glucose transport 1 (GLUT1), one of the receptor molecules of the HTLV-1 virus, and the receptor molecule required for HTLV-1 fusion and entry. We postulated that Tax-1 alters GLUT1 localization via its interaction with SNX27. We demonstrate that over expression of Tax-1 in cells causes a reduction of GLUT1 on the plasma membrane. Furthermore, we show that knockdown of SNX27 results in increased virion release and decreased HTLV-1 infectivity. Collectively, we demonstrate the first known mechanism by which HTLV-1 regulates a receptor molecule post-infection.

CD147 and Cyclooxygenase Expression in Feline Oral Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (OSCC) is a highly invasive form of cancer in cats. In human OSCC, cluster of differentiation 147 (CD147) contributes to inflammation and tumor invasiveness. CD147 is a potential therapeutic target, but the expression of CD147 in feline OSCC has not been examined. Immunohistochemistry was used to determine if cyclooxygenase 2 (COX-2) and CD147 expression in feline OSCC biopsies was coordinated. Tumor cells were more likely to express COX-2 (22/43 cases or 51%) compared to stroma (8/43 or 19%) and adjacent oral epithelium (9/31 cases or 29%) (p < 0.05). CD147 was also more likely to occur in tumor cells compared to stroma and adjacent mucosa, with 21/43 (49%) of cases having >50% tumor cells with mild or moderate CD147 expression, compared to 9/28 (32%) in adjacent epithelium and only 5/43 (12%) in adjacent stroma (p < 0.05). In feline OSCC cell lines (SCCF1, SCCF2, and SCCF3), CD147 gene expression was more consistently expressed compared to COX-2, which was 60-fold higher in SCCF2 cells compared to SCCF1 cells (p < 0.05). CD147 expression did not correlate with COX-2 expression and prostaglandin E2 (PGE2) secretion, indicating that they may be independently regulated. CD147 potentially represents a novel therapeutic target for the treatment of feline OSCC and further study of CD147 is warranted.

Development of an orthotopic canine prostate cancer model expressing human GRPr.

BACKGROUND: Ace-1 canine prostate cancer cells grow orthotopically in cyclosporine immunosuppressed laboratory beagles. We previously transfected (human Gastrin-Releasing Peptide Receptor, huGRPr) into Ace-1 cells and demonstrated receptor-targeted NIRF imaging with IR800-G-Abz4-t-BBN, an agonist to huGRPr. Herein, we used the new cell line to develop the first canine prostate cancer model expressing a human growth factor receptor. METHODS: Dogs were immunosuppressed with cyclosporine, azathioprine, prednisolone, and methylprednisolone. Their prostate glands were implanted with Ace-1huGRPr cells. The implantation wounds were sealed with a cyanoacrylic adhesive to prevent extraprostatic tumor growth. Intraprostatic tumors grew in 4-5 week. A lobar prostatic artery was then catheterized via the carotid artery and 25-100 nmol IR800-Abz4-t-BBN was infused in 2 mL followed by euthanasia in dogs 1-2, and recovery for 24 h before euthanasia in dogs 3-6. Excised tissues were imaged optically imaged, and histopathology performed. RESULTS: Dog1 grew no tumors with cyclosporine alone. Using the four drug protocol, Dogs 2-6 grew abundant 1-2 mm intracapsular and 1-2 cm intraglandular tumors. Tumors grew >5 cm when the prostate cancer cells became extracapsular. Dogs 4-6 with sealed prostatic capsule implantation sites had growth of intracapsular and intraglandular tumors and LN metastases at 5 weeks. High tumor to background BPH signal in the NIRF images of sectioned prostate glands resulted from the 100 nmol dose (∼8 nmol/kg) in dogs 2-4 and 50 nmol dose in dog 5, but not from the 25 nmol dose in Dog 6. Imaging of mouse Ace-1huGRPr tumors required an intravenous dose of 500 nmol/kg body wt. A lymph node that drained the prostate gland was detectable in Dog 4. Histologic findings confirmed the imaging data. CONCLUSION: Ace-1huGRPr cells created viable, huGRPr-expressing tumors when implanted orthotopically into immune-suppressed dogs. Local delivery of an imaging agent through the prostatic artery allowed a very low imaging dose, suggesting that therapeutic agents could be used safely for treatment of early localized intraglandular prostate cancer as adjuvant therapy for active surveillance or focal ablation therapies, or for treating multifocal intraglandular disease where focal ablation therapies are not indicated or ineffective.

Discovery and characterization of the feline miRNAome.

The domestic cat is an important human companion animal that can also serve as a relevant model for ~250 genetic diseases, many metabolic and degenerative conditions, and forms of cancer that are analogous to human disorders. MicroRNAs (miRNAs) play a crucial role in many biological processes and their dysregulation has a significant impact on important cellular pathways and is linked to a variety of diseases. While many species already have a well-defined and characterized miRNAome, miRNAs have not been carefully studied in cats. As a result, there are no feline miRNAs present in the reference miRNA databases, diminishing the usefulness of medical research on spontaneous disease in cats for applicability to both feline and human disease. This study was undertaken to define and characterize the cat miRNAome in normal feline tissues. High-throughput sequencing was performed on 12 different normal cat tissues. 271 candidate feline miRNA precursors, encoding a total of 475 mature sequences, were identified, including several novel cat-specific miRNAs. Several analyses were performed to characterize the discovered miRNAs, including tissue distribution of the precursors and mature sequences, genomic distribution of miRNA genes and identification of clusters, and isomiR characterization. Many of the miRNAs were regulated in a tissue/organ-specific manner.

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.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

BACKGROUND: The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. METHODS: The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. RESULTS: Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and ß-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. CONCLUSION: These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc.

A human GRPr-transfected Ace-1 canine prostate cancer model in mice.

BACKGROUND: A versatile drug screening system was developed to simplify early targeted drug discovery in mice and then translate readily from mice to a dog prostate cancer model that more fully replicates the features of human prostate cancer. METHODS: We stably transfected human cDNA of the GRPr bombesin (BBN) receptor subtype to canine Ace-1 prostate cancer cells (Ace-1(huGRPr) ). Expression was examined by (125) I-Tyr(4) -BBN competition, calcium stimulation assay, and fluorescent microscopy. A dual tumor nude mouse xenograft model was developed from Ace-1(CMV) (vector transfected Ace-1) and Ace-1(huGRPr) cells. The model was used to explore the in vivo behavior of two new IRDye800-labeled GRPr binding optical imaging agents: 800-G-Abz4-t-BBN, from a GRPr agonist peptide, and 800-G-Abz4-STAT, from a GRPr antagonist peptide, by imaging the tumor mice and dissected organs. RESULTS: Both agents bound Ace-1(huGRPr) and PC-3, a known GRPr-expressing human prostate cancer cell line, with 4-13 nM IC50 against (125) I-Tyr(4) -BBN, but did not bind Ace-1(CMV) cells (vector transfected). Binding was blocked by bombesin. Ca(2+) activation assays demonstrated that Ace-1(huGPRr) expressed biologically active GRPr. Both Ace-1 cell lines grew in the flanks of 100% of the nude mice and formed tumors of ∼0.5 cm diameter in 1 week. In vivo imaging of the mice at 800 nm emission showed GRPr+: GRPr- tumor signal brighter by a factor of two at 24 h post IV administration of 10 nmol of the imaging agents. Blood retention (4-8% ID at 1 h) was greater by a factor >10 and cumulative urine accumulation (28-30% at 4 h) was less by a factor 2 compared to a radioactive analog of the t-BBN containing agent, (177) LuAMBA, probably due to binding to blood albumin, which we confirmed in a mouse serum assay. CONCLUSIONS: The dual tumor Ace-1(CMV) /Ace-1(huGRPr) model system provides a rapid test of specific to nonspecific binding of new GRPr avid agents in a model that will extend logically to the known Ace-1 orthotopic canine prostate cancer model. Prostate 76:783-795, 2016. © 2016 Wiley Periodicals, Inc.

Animal models of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the most common oral cancer worldwide. Local bone invasion into the maxilla or mandible and metastasis to regional lymph nodes often result in a poor prognosis, decreased quality of life and shortened survival time for HNSCC patients. Poor response to treatment and clinical outcomes are the major concerns in this aggressive cancer. Multiple animal models have been developed to replicate spontaneous HNSCC and investigate genetic alterations and novel therapeutic targets. This review provides an overview of HNSCC as well as the traditional animal models used in HNSCC preclinical research. The value and challenges of each in vivo model are discussed. Similarity between HNSCC in humans and cats and the possibility of using spontaneous feline oral squamous cell carcinoma (FOSCC) as a model for HNSCC in translational research are highlighted.

p16, pRb, and p53 in Feline Oral Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a highly aggressive head and neck cancer in cats, but the molecular pathogenesis of this cancer is still uncertain. In this study, p16, p53, and pRb proteins were detected and quantified by immunohistochemistry in forty-three FOSCC primary tumors and three FOSCC xenografts. p16 mRNA levels were also measured in three FOSCC cell lines (SCCF1, F2, and F3), which were consistent with their p16 immunoreactivity. Feline SCCF1 cells had very high levels of p16 protein and mRNA (55-fold greater) compared to SCCF2 and F3. A partial feline p16 cDNA sequence was amplified and sequenced. The average age of cats with FOSCC with high p16 immunoreactivity was significantly lower than the average age in the low p16 group. Eighteen of 43 (42%) FOSCCs had low p16 intensity, while 6/43 (14%) had high p16 immunoreactivity. Feline papillomavirus L1 (major capsid) DNA was not detected in the SCC cell lines or the FOSCCs with high p16 immunostaining. Five of 6 (83%) of the high p16 FOSCC had low p53, but only 1/6 (17%) had low pRb immunoreactivity. In summary, the staining pattern of p16, p53, and pRb in FOSCC was different from human head and neck squamous cell carcinoma and feline cutaneous squamous cell carcinoma. The majority of FOSCCs have low p16 immunostaining intensity, therefore, inactivation of CDKN2A is suspected to play a role in the pathogenesis of FOSCC. A subset of FOSCCs had increased p16 protein, which supports an alternate pathogenesis of cancer in these cats.

Deletion of the nuclear localization sequence and C-terminus of parathyroid hormone-related protein decreases osteogenesis and chondrogenesis but increases adipogenesis and myogenesis in murine bone marrow stromal cells.

The N-terminus of parathyroid hormone-related protein regulates bone marrow stromal cell differentiation. We hypothesized that the nuclear localization sequence and C-terminus are involved. MicroRNA and gene expression analyses were performed on bone marrow stromal cells from mice lacking the nuclear localization sequence and C-terminus (PthrpΔ/Δ ) and age-matched controls. Differentiation assays with microRNA, cytochemical/histologic/morphologic, protein, and gene expression analyses were performed. PthrpΔ/Δ bone marrow stromal cells are anti-osteochondrogenic, pro-adipogenic, and pro-myogenic, expressing more Klf4, Gsk-3ß, Lif, Ct-1, and microRNA-434 but less ß-catenin, Igf-1, Taz, Osm, and microRNA-22 (p ⩽ 0.024). PthrpΔ/Δ osteoblasts had less mineralization, osteocalcin, Runx2, Osx, Igf-1, and leptin (p ⩽ 0.029). PthrpΔ/Δ produced more adipocytes, Pparγ, and aP2, but less Lpl (p ⩽ 0.042). PthrpΔ/Δ cartilage pellets were smaller with less Sox9 and Pth1r, but greater Col2a1 (p ⩽ 0.024). PthrpΔ/Δ produced more myocytes, Des, and Myog (p ⩽ 0.021). MicroRNA changes supported these findings. In conclusion, the nuclear localization sequence and C-terminus are pro-osteochondrogenic, anti-adipogenic, and anti-myogenic.

Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo.

BACKGROUND: In 2012, over 240,000 men were diagnosed with prostate cancer and over 28,000 died from the disease. Animal models of prostate cancer are vital to understanding its pathogenesis and developing therapeutics. Canine models in particular are useful due to their similarities to late-stage, castration-resistant human disease with osteoblastic bone metastases. This study established and characterized a novel canine prostate cancer cell line that will contribute to the understanding of prostate cancer pathogenesis. METHODS: A novel cell line (Probasco) was derived from a mixed breed dog that had spontaneous prostate cancer. Cell proliferation and motility were analyzed in vitro. Tumor growth in vivo was studied by subcutaneous, intratibial, and intracardiac injection of Probasco cells into nude mice. Tumors were evaluated by bioluminescent imaging, Faxitron radiography, µCT, and histology. RT-PCR and genome-wide DNA copy number profiling were used to characterize the cell line. RESULTS: The Probasco cells grew in vitro (over 75 passages) and were tumorigenic in nude mice. Probasco cells expressed high levels of BMP2, CDH1, MYOF, FOLH1, RUNX2, and SMAD5 modest CXCL12, SLUG, and BMP, and no PTHrP mRNA. Following intracardiac injection, Probasco cells metastasized primarily to the appendicular skeleton, and both intratibial and intracardiac injections produced osteoblastic tumors in bone. Comparative genomic hybridization demonstrated numerous DNA copy number aberrations throughout the genome, including large losses and gains in multiple chromosomes. CONCLUSIONS: The Probasco prostate cancer cell line will be a valuable model to investigate the mechanisms of prostate cancer pathogenesis and osteoblastic bone metastases.

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. METHODS: Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. RESULTS: Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. CONCLUSIONS: The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.

AU-rich elements in the 3'-UTR regulate the stability of the 141 amino acid isoform of parathyroid hormone-related protein mRNA.

We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-ß. In order to understand how PTHrP mRNA is stabilized by TGF-ß, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3'-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3'-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA.