Maran, Avudaiappan

10531 Background: Osteosarcoma (OS) is a clinically challenging primary malignant bone tumor that has a high rateof local recurrence and metastasis. Disease free survival has been reported to be as low as 19% despite contemporary chemotherapy regimens and surgery. Wnt signaling is known to play a role in osteogenesis and cancer pathogenesis, therefore is a promising target for therapeutic intervention in OS. Secreted Frizzled Related Proteins (sFRPs), Dickkopf-1 (DKK1), and Sclerostin (SOST) are secreted Wnt inhibitor proteins that regulate Wnt signaling in bone. This study investigates the activity of sFRP3, DKK1, and SOST in serum from OS patients with the goal of developing therapeutic Wnt inhibitors and clinically useful biomarkers. Methods: Enzyme linked immunosorbent assayswere used to quantify sFRP3, DKK1 and SOST levels in human serum (n=40 pairs; 20 OS patients, 20 aged matched non-diseased controls). Patients were stratified into 2 age groups: >35 (n=26 pairs) and <30 (n=14 pairs) years of age. Clinical data from the medical records was correlated with experimental results. Using a Wilcoxon signed rank test, a p–value <0.05 was significant. Results: sFRP3 levels were significantly decreased in 55% (22/40) of diseased samples compared to 20% (8/40) of the non-diseased controls (p= 0.007). DKK1 levels were elevated in 40% (16/40) of OS samples compared to 30% (12/40) of the non-diseased controls (p= 0.08). SOST levels were increased in 42.5% (17/40) of diseased samples compared to 32.5% (13/40) of the non-diseased controls (p= 0.38). When stratified by age, the younger patients showed a statistically significant decrease in sFRP3 expression (p<0.001). Serum levels of sFRP3, DKK1, and SOST were not statistically significant between metastatic and non-metastatic OS. Conclusions: Serum sFRP3 levels were significantly decreased in OS patients, a difference that was even more pronounced in the pediatric strata. Since sFRP3 is differentially expressed in patient serum it has potential as a clinical biomarker for initial tumor diagnosis and early detection of recurrence. Decreased sFRP3 expression may be responsible for the up regulation of Wnt signaling in OS. Wnt inhibitors such as sFRP3 have potential as therapeutic agents in OS warranting further investigation.

Accurate clinical evaluation of renal osteodystrophy (ROD) is currently accomplished using invasive in vivo transiliac bone biopsy, followed by in vitro histomorphometry. In this study, we demonstrate that an alternative method for ROD assessment is through a fast, label-free Raman recording of multiple biomarkers combined with computational analysis for predicting the minimally required number of spectra for sample classification at defined accuracies. Four clinically relevant biomarkers: the mineral-to-matrix ratio, the carbonate-to-matrix ratio, phenylalanine, and calcium contents were experimentally determined and simultaneously considered as input to a linear discriminant analysis (LDA). Additionally, sample evaluation was performed with a linear support vector machine (LSVM) algorithm, with a 300 variable input. The computed probabilities based on a single spectrum were only marginally different (~80% from LDA and ~87% from LSVM), both providing an unacceptable classification power for a correct sample assignment. However, the Type I and Type II assignment errors confirm that a relatively small number of independent spectra (7 spectra for Type I and 5 spectra for Type II) is necessary for a p < 0.05 error probability. This low number of spectra supports the practicality of future in vivo Raman translation for a fast and accurate ROD detection in clinical settings.

Defining the pathogenesis of renal osteodystrophy (ROD) and its treatment efficacy are difficult, since many factors potentially affect bone quality. In this study, confocal Raman microscopy and parallel statistical analysis were used to identify differences in bone composition between healthy and ROD bone tissues through direct visualization of three main compositional parametric ratios, namely, calcium content, mineral-to-matrix, and carbonate-to-matrix. Besides the substantially lower values found in ROD specimens for these representative ratios, an obvious accumulation of phenylalanine is Raman spectroscopically observed for the first time in ROD samples and reported here. Thus, elevated phenylalanine could also be considered as an indicator of the disease. Since the image results are based on tens of thousands of spectra per sample, not only are the average ratios statistically significantly different for normal and ROD bone, but the method is clearly powerful in distinguishing between the two types of samples. Furthermore, the statistical outcomes demonstrate that only a relatively small number of spectra need to be recorded in order to classify the samples. This work thus opens the possibility of future development of in vivo Raman sensors for assessment of bone structure, remodeling, and mineralization, where different biomarkers are simultaneously detected with unprecedented accuracy.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1–32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.

ABSTRACTOsteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti‐tumor compound, 2‐methoxyestradiol (2‐ME) on Wnt antagonist frizzled‐related protein b (Frzb), also known as secreted frizzled‐related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2‐ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2‐ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β‐catenin, and blocking β‐catenin‐mediated Wnt activation in osteosarcoma cells. 2‐ME‐mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2‐ME‐induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2‐ME‐mediated anti‐tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497–1504, 2017. © 2016 Wiley Periodicals, Inc.

Systematic review of experimental and clinical data on the use of curcumin in the treatment of osteosarcoma is presented. The current status of curcumin’s therapeutic potential against bone cancer is analyzed in regard to using polymeric micelles (including recently developed invertible, responsive, micelles) as a platform for curcumin delivery to treat osteosarcoma. The potential of micellar assemblies from responsive macromolecules in a controlled delivery of curcumin to osteosarcoma cells, and the release using a new inversion mechanism is revealed.

Abstract Purpose: The objective of this study is to characterize the function of porcupine inhibitors in osteosarcoma and to determine the contribution of the porcupine enzyme to osteosarcoma cell growth. Each year, approximately 1000 children and adults in the United States are diagnosed with osteosarcoma (OS), the most common primary bone malignancy among children. Although overall survival is relatively good in OS patients, the need for aggressive surgery (often involving limb amputation) to prevent relapse significantly impacts the quality of life of survivors. OS has been well characterized to display aberrantly activated Wnt signaling. The necessity for active Wnt signaling pathways in OS patient tissue and cultured cell lines has been well established. Recently, a group of Wnt pathway inhibitors targeting the porcupine enzyme have been developed and used in treatment of pancreatic adenocarcinoma, BRAF mutant colorectal cancer and other tumors reported to exhibit aberrant Wnt signaling. Porcupine is a membrane bound O-acyl transferase which lipid-modifies the Wnt proteins prior to their secretion. This processing step regulates their release and paracrine or autocrine activity in stimulating both canonical and non-canonical Wnt signaling. These drugs have proven effective in initial studies which have applied them in vitro, in mouse models of Wnt- dependent cancers, and a recent phase 1 clinical trial. Our data show the first report of increased porcupine enzyme expression in human OS in vitro cell lines, compared to primary human osteoblasts. These findings have been confirmed by quantitative real-time PCR (qRT-PCR) analysis of PPN mRNA expression. Surprisingly, we found that inhibition of the porcupine enzyme with newly developed porcupine inhibitors significantly increased OS cell viability. While we observed only slight decreases in canonical Wnt pathway signaling with PPN inhibitor treatment (as measured by TOPflash reporter assay), we found that both NFATc1 and Cyclin D1 mRNA expression was upregulated. These data indicate that treatment with PPN inhibitors may stimulate a pro-tumorigenic response that is mediated primarily through non-canonical Wnt signaling. Continuing work is being done to establish the mechanism of action of these drugs, as well as unique roles that PPN-mediated non-canonical Wnt signaling may play in promoting OS cell growth. In conclusion, this study is aimed to provide insight into the function of a group of novel Wnt-targeting therapies in osteosarcoma, a disease which is well established to exhibit aberrant Wnt signaling. We aim to utilize these insights to rationally exploit Wnt signaling as a therapeutic measure for osteosarcoma. Methods: Western blotting was performed according to accepted methods using BioRad Criterion™ 10% gel and an antibody polyclonal to known porcupine splice variants (anti-rabbit) obtained from GeneTex. Cell viability was determined by CellTiter 96 ® (Promega) proliferation assay. C59 (Novartis) was purchased from Cayman Chemical and reconstituted in DMSO. Quantitative PCR performed using an Applied Biosystems ABI 7900HT Fast Real Time PCR system. FuGENE® HD utilized for transfection of TOPflash components. Citation Format: Carl T. Gustafson, Avudai Maran, Michael J. Yaszemski. Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B11.