Alterations in Sod2-Induced Oxidative Stress Affect Endocrine Cancer Progression.

Context: Although important advances have been made in understanding the genetics of endocrine tumors, cellular physiology is relatively understudied as a determinant of tumor behavior. Oxidative stress and reactive oxygen species are metabolic factors that may affect tumor behavior, and these are, in part, controlled by manganese-dependent superoxide dismutase (MnSod), the mitochondrial superoxide dismutase (encoded by SOD2). Objective: We sought to understand the role of MnSod in the prognosis of aggressive human endocrine cancers and directly assessed the effect of MnSod under- or overexpression on tumor behavior, using established mouse thyroid cancer models. Methods: We performed transcriptome analysis of human and mouse models of endocrine cancer. To address the role of Sod2 in endocrine tumors, we introduced a Sod2 null allele or a transgenic Sod2 overexpression allele into mouse models of benign thyroid follicular neoplasia or aggressive, metastatic follicular thyroid cancer (FTC) and monitored phenotypic changes in tumor initiation and progression. Results: In the thyroid, SOD2/Sod2 was downregulated in FTC but not papillary thyroid cancer. Reduced expression of SOD2 was correlated with poorer survival of patients with aggressive thyroid or adrenal cancers. In mice with benign thyroid tumors, Sod2 overexpression increased tumor burden. In contrast, in mice with aggressive FTC, overexpression of Sod2 reduced tumor proliferation and improved mortality rates, whereas its deficiency enhanced tumor growth. Conclusion: Overall, our results indicate that SOD2 has dichotomous roles in cancer progression and acts in a context-specific manner.

Deletion of Rap1b, but not Rap1a or Epac1, Reduces Protein Kinase A-Mediated Thyroid Cancer.

BACKGROUND: Thyroid cancer is an emerging health problem in the United States and worldwide. With incidence rates of thyroid cancer rapidly rising, the need to develop new treatment options is becoming a priority, and understanding the molecular mechanisms of this disease is crucial to furthering these efforts. Thyroid growth is driven by the TSH/cAMP/PKA signaling pathway, and it has previously been shown that activation of PKA through genetic ablation of the regulatory subunit Prkar1a (Prkar1a KO) is sufficient to cause follicular thyroid cancer in mouse models. cAMP also activates the Epac proteins and their downstream effectors, Rap1a and Rap1b. METHODS: Previously, the authors' laboratory generated a mouse model of follicular thyroid cancer by conferring thyroid-specific deletion of Prkar1a (R1a-TpoKO). To probe the roles of other components of the PKA signaling system in the development of thyroid cancer, this study deleted Rap1 and Epac1 in the setting of the Prkar1a knockout. RESULTS: Deletion of Rap1 significantly decreases thyroid size and cancer incidence in Prkar1a KO thyroids. Further, isoform-specific ablation of Rap1a and Rap1b implicates Rap1b as the downstream effector of PKA during thyroid carcinogenesis. In vivo modeling provides definitive evidence that Epac1 plays little role in thyroid proliferation and is dispensable for thyroid carcinogenesis arising from the deletion of Prkar1a. CONCLUSIONS: This study demonstrate that PKA signaling to Rap1b is a key signaling node for follicular thyroid carcinogenesis, while Epac1 activity is not required for tumor development. This work sheds new light on the pathways involved in FTC development and identifies a possible target for the development of new therapies in the treatment of FTC.

Sdhd ablation promotes thyroid tumorigenesis by inducing a stem-like phenotype.

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro, human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia.

Ovariectomy-Induced Osteoporosis Does Not Impact Fusion Rates in a Recombinant Human Bone Morphogenetic Protein-2-Dependent Rat Posterolateral Arthrodesis Model.

Study Design Randomized, controlled animal study. Objective Recombinant human bone morphogenetic protein-2 (rhBMP-2) is frequently utilized as a bone graft substitute in spinal fusions to overcome the difficult healing environment in patients with osteoporosis. However, the effects of estrogen deficiency and poor bone quality on rhBMP-2 efficacy are unknown. This study sought to determine whether rhBMP-2-induced healing is affected by estrogen deficiency and poor bone quality in a stringent osteoporotic posterolateral spinal fusion model. Methods Aged female Sprague-Dawley rats underwent an ovariectomy (OVX group) or a sham procedure, and the OVX animals were fed a low-calcium, low-phytoestrogen diet. After 12 weeks, the animals underwent a posterolateral spinal fusion with 1 µg rhBMP-2 on an absorbable collagen sponge. Representative animals were sacrificed at 1 week postoperative for alkaline phosphatase (ALP) and osteocalcin serum analyses. The remaining animals underwent radiographs 2 and 4 weeks after surgery and were subsequently euthanized for fusion analysis by manual palpation, micro-computed tomography (CT) imaging, and histologic analysis. Results The ALP and osteocalcin levels were similar between the control and OVX groups. Manual palpation revealed no significant differences in the fusion scores between the control (1.42 ± 0.50) and OVX groups (1.83 ± 0.36; p = 0.07). Fusion rates were 100% in both groups. Micro-CT imaging revealed no significant difference in the quantity of new bone formation, and histologic analysis demonstrated bridging bone across the transverse processes in fused animals from both groups. Conclusions This study demonstrates that estrogen deficiency and compromised bone quality do not negatively influence spinal fusion when utilizing rhBMP-2, and the osteoinductive capacity of the growth factor is not functionally reduced under osteoporotic conditions in the rat. Although osteoporosis is a risk factor for pseudarthrosis/nonunion, rhBMP-2-induced healing was not inhibited in osteoporotic rats.

Effect of recombinant human bone morphogenetic protein-2 on a novel lung cancer spine metastasis model in rodents.

Lung cancer is the second most prevalent cancer. Spinal metastases are found in 30-90% of patients with death attributed to cancer. Due to bony destruction caused by metastases, surgical intervention is often required to restore spinal alignment and stability. While some research suggests that BMP-2 may possess tumorigenic effects, other studies show possible inhibition of cancer growth. Thirty-six athymic rats underwent intraosseous injection of lung adenocarcinoma cells into the L5 vertebral body. Cells were pre-treated with vehicle control (Group A) or rhBMP-2 (Group B) prior to implantation. At 4 weeks post-implantation, in vivo bioluminescent imaging (BLI) was performed to confirm presence of tumor and quantify signal. Plain radiographs and microComputed Tomography (microCT) were employed to establish and quantitate osteolysis. Histological analysis characterized pathologic changes in the vertebral body. At 4 weeks post-implantation, BLI showed focal signal in the L5 vertebral body in 93% of Group A animals and 89% of Group B animals. Average tumor burden by BLI radiance was 7.43 × 10(3) p/s/cm(2) /sr (Group A) and 1.11 × 10(4) p/s/cm(2) /sr (Group B). Radiographs and microCT demonstrated osteolysis in 100% of animals showing focal BLI signal. MicroCT demonstrated significant bone loss in both groups compared to age-matched controls but no difference between study groups. Histological analysis confirmed tumor invasion in the L5 vertebral body. These findings provide a reliable in vivo model to study isolated spinal metastases from lung cancer. Statement of Clinical Significance: The data support the notion that exposure to rhBMP-2 does not promote the growth of A549 lung cancer spine lesions. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1274-1281, 2016.

Mouse models of thyroid cancer: A 2015 update.

Thyroid cancer is the most common endocrine neoplasm, and its rate is rising at an alarming pace. Thus, there is a compelling need to develop in vivo models which will not only enable the confirmation of the oncogenic potential of driver genes, but also point the way towards the development of new therapeutics. Over the past 20 years, techniques for the generation of mouse models of human diseases have progressed substantially, accompanied by parallel advances in the genetics and genomics of human tumors. This convergence has enabled the development of mouse lines carrying mutations in the genes that cause thyroid cancers of all subtypes, including differentiated papillary and follicular thyroid cancers, poorly differentiated/anaplastic cancers, and medullary thyroid cancers. In this review, we will discuss the state of the art of mouse modeling of thyroid cancer, with the eventual goal of providing insight into tumor biology and treatment.

Dioxin Exposure Impairs BMP-2-Mediated Spinal Fusion in a Rat Arthrodesis Model.

BACKGROUND: Cigarette smoking inhibits bone-healing and leads to increased rates of pseudarthrosis. However, the mechanisms behind these effects are controversial. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)--a cigarette smoke constituent and potent activator of the aryl hydrocarbon receptor (Ahr)--negatively impacts bone quality and osteoblast differentiation. We hypothesized that activation of the Ahr by dioxin would inhibit bone morphogenetic protein (BMP)-2-mediated spinal fusion in a rat arthrodesis model. METHODS: Female Long-Evans rats were pretreated with dioxin or vehicle in six weekly doses, followed by bilateral posterior lumbar spinal fusion across the L4-L5 transverse processes using recombinant human BMP (rhBMP)-2. Treatments continued until sacrifice at four weeks postoperatively. A third group was treated with dioxin for six weeks, followed by a recovery period of four elimination half-lives to assess the reversible effects of dioxin exposure on spinal fusion capacity. Bone formation and fusion capacity were evaluated using fusion scoring, radiography, micro-computed tomography, and histologic analysis. RESULTS: Fusion scores for dioxin-treated and dioxin-recovery rats were significantly lower than those for controls. Although fusion rates were also significantly reduced in dioxin-treated animals relative to controls (50% versus 100%, respectively), rates were not significantly reduced in dioxin-recovery animals (80%). CONCLUSIONS: Dioxin treatment significantly inhibited spinal fusion in a rat arthrodesis model, and a prolonged cessation of dioxin exposure facilitated only a partial recovery of bone-healing capacity. This finding indicates that, although the effects of dioxin are persistent, an extended recovery from exposure could potentially restore bone regeneration in vivo. CLINICAL RELEVANCE: Development of a pharmacologic agent that reduces the adverse effects of cigarette smoke on bone-healing could prove useful to orthopaedic surgeons. Since dioxin and other similar cigarette smoke toxins exert their effects through Ahr pathway activation, the receptor represents a potential therapeutic target to improve spinal fusion rates in patients who smoke.

Gel scaffolds of BMP-2-binding peptide amphiphile nanofibers for spinal arthrodesis.

Peptide amphiphile (PA) nanofibers formed by self-assembly can be customized for specific applications in regenerative medicine through the use of molecules that display bioactive signals on their surfaces. Here, the use of PA nanofibers with binding affinity for the bone promoting growth factor BMP-2 to create a gel scaffold for osteogenesis is reported. With the objective of reducing the amount of BMP-2 used clinically for successful arthrodesis in the spine, amounts of growth factor incorporated in the scaffolds that are 10 to 100 times lower than that those used clinically in collagen scaffolds are used. The efficacy of the bioactive PA system to promote BMP-2-induced osteogenesis in vivo is investigated in a rat posterolateral lumbar intertransverse spinal fusion model. PA nanofiber gels displaying BMP-2-binding segments exhibit superior spinal fusion rates relative to controls, effectively decreasing the required therapeutic dose of BMP-2 by 10-fold. Interestingly, a 42% fusion rate is observed for gels containing the bioactive nanofibers without the use of exogenous BMP-2, suggesting the ability of the nanofiber to recruit endogenous growth factor. Results obtained here demonstrate that bioactive biomaterials with capacity to bind specific growth factors by design are great targets for regenerative medicine.

A comparative evaluation of factors influencing osteoinductivity among scaffolds designed for bone regeneration.

Due to differing compositions, synthetic scaffolds developed for bone regeneration vary widely in efficacy. To quantify the impact of such differences on osteoinductivity, numerous parameters were examined. Absorbable collagen sponge (ACS), three ceramic-based carriers (#1-3) of varying compositions, mineralized allograft chips, and an experimental phosphoserine-rich nanofiber scaffold [S(P) gel] were compared in their ability to promote cell adhesion, proliferation/survival, growth factor binding/release, and osteogenic gene expression. Human preosteoblasts were found to adhere most efficiently to the S(P) gel, and the growth/survival was greatest on the S(P) and ACS scaffolds, with minimal growth seen on the allograft and Ceramic #3. In bone morphogenetic protein-2 (BMP-2) binding/release assays, ACS demonstrated a burst release pattern, whereas the allograft and the ceramics inefficiently released BMP-2. The S(P) gel showed the most ideal rates of growth factor binding and release. QPCR analyses showed significant differences in the CXCL12, CXCR4, and RANKL transcripts among the cells grown on these various scaffolds. Although some scaffolds showed an advantage over others in individual parameters, the nanofiber gel appears to provide the optimal balance in the factors important to osteoinductivity evaluated here.