Purpose Notch1, a trans-membrane receptor, has been recently shown to aid in the determination of thyroid cell fate associated with tumorigenesis. The present study aimed to investigate the clinical relevance of Notch1 and its role in the regulation of differentiated thyroid cancer (DTC) behavior. Experimental design We examined Notch1 expression level and its relationship with clinicopathologic features and outcomes of DTC. Notch1 intracellular domain (NICD) was further characterized both in vitro and in vivo by gain-of-function assays using an inducible system. Results Notch1 expression levels were down-regulated in primary DTC tissue samples compared with contralateral non-tumor and benign thyroid tissues. Decreased Notch1 expression in DTC was associated with advanced patient age (p=0.032) and the presence of extrathyroidal invasion (p=0.005). Patients with lower Notch1 expression had a significantly higher recurrence rate (p=0.038). Restoration of NICD in a stably doxycycline-inducible metastatic DTC cell line reduced cell growth and migration profoundly. Using an orthotopic thyroid cancer model, NICD induction significantly reduced the growth of the primary thyroid tumor and inhibited the development of lung metastasis. SERPINE1 was discovered by microarray as the most significant gene down-regulated by NICD. Further validation showed that induction of NICD reduced SERPINE1 expression in a dose-dependent manner while restoration of a relative higher level of SERPINE1was observed with NICD back to minimal level. Additionally, SERPINE1 knock-down inhibited DTC cell migration. Conclusions Notch1 regulates the aggressive phenotypes of DTC, which could be mediated by SERPINE1 inhibition. Notch1/SERPINE1 axis warrants further investigation as a novel therapeutic target for advanced DTC.

Background Anaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with anti-proliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. Methods Human-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. Results TDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly ADP ribose polymerase (PARP) were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently and the anti-apoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase following TDP-A treatment. Conclusion TDP-A induces a notable dose- and time-dependent anti-proliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

Importance Parathyroidectomy offers the only cure for primary hyperparathyroidism (PHPT), but today only 50% of PHPT patients are referred for surgery, in large part because the condition is widely under-recognized. PHPT diagnosis can be especially challenging with mild biochemical indices. Machine learning (ML) is a collection of methods in which computers build predictive algorithms based on labeled examples. Objective With the aim of facilitating diagnosis, we tested the ability of ML to distinguish PHPT from normal physiology using clinical and laboratory data. Design This is a retrospective cohort study using a labeled training set and 10-fold cross-validation to evaluate algorithm accuracy. Measures of accuracy included area under the ROC curve, precision (sensitivity), and positive and negative predictive value. Several different ML algorithms and ensembles of algorithms were tested using the Weka platform. Setting 3 high-volume endocrine surgery programs Participants Among 11,830 patients managed surgically from March, 2001 to August 2013, 6,777 underwent parathyroidectomy for PHPT, and 5,053 control patients without PHPT underwent thyroidectomy. Main Outcomes and Measures Test-set accuracies for ML models were determined using 10-fold cross-validation. Age, gender, preoperative calcium, phosphate, PTH, Vitamin D, and creatinine were defined as potential predictors of PHPT. Mild PHPT was defined as PHPT with normal preoperative calcium or PTH levels. Results After testing a variety of ML algorithms, Bayesian network models proved most accurate, correctly classifying 95.2% of all PHPT patients (area under ROC=0.989). Omitting PTH from the model did not significantly reduce the accuracy (area under ROC = 0.985). However, in mild disease cases, the Bayesian network model correctly classified 71.1% of patients with normal calcium and 92.1% with normal PTH levels preoperatively. Bayesian networking + AdaBoost improved the accuracy to 97.2% correctly classified (area under ROC=0.994) cases, and 91.9% of PHPT patients with mild disease. This was significantly improved relative to Bayesian networking alone (p<0.0001). Conclusions and Relevance ML can accurately diagnose PHPT without human input, even in mild disease. Incorporation of this tool into electronic medical record systems may greatly aid in recognition of this under-diagnosed disorder.

Purpose: The PI3K/Akt/mTOR prosurvival pathway is frequently up-regulated in soft tissue sarcoma. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, have recently shown clinical benefit in soft tissue sarcoma, and mTOR inhibition has also been associated with radiosensitization of carcinoma and endothelial cells. This study tested the hypothesis that rapamycin radiosensitizes soft tissue sarcoma and endothelial cells in vitro and in vivo through the inhibition of mTOR. Experimental Design: Colony formation assays were done to determine the radiosensitizing properties of rapamycin on three human soft tissue sarcoma cell lines (SK-LMS-1, SW-872, and HT-1080) and human dermal microvascular endothelial cells (HDMEC). The functional effects of rapamycin and radiation on the endothelial compartment were evaluated with microvascular sprouting assays. The in vivo radiosensitizing activity of rapamycin was assessed with s.c. SK-LMS-1 nude mice xenografts treated with concurrent daily rapamycin, radiation, or both for three weeks. Results: In vitro radiosensitization was shown in all three soft tissue sarcoma cell lines with minimally cytotoxic doses of rapamycin. SK-LMS-1 xenografts displayed significant tumor growth delay with rapamycin and radiation compared with either treatment alone. Radiation resulted in transient increased mTOR function, whereas rapamycin abolished this signaling in irradiated and unirradiated samples. In HDMEC, rapamycin and radiation reduced microvessel sprouting, but did not alter colony formation. Conclusions: Minimally cytotoxic concentrations of rapamycin inhibited the mTOR cascade in culture and in vivo while radiosensitizing soft tissue sarcoma, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, rapamycin produced potent radiosensitization of soft tissue sarcoma xenografts. Clinical trials combining rapamycin and radiotherapy in soft tissue sarcoma are warranted.

BACKGROUND Thyroid tumorigenesis is characterized by a progressive loss of differentiation exhibited by a range of disease variants. The Notch(1-4) receptor family regulates developmental progression in both normal and cancerous tissues. We sought to characterize the third Notch isoform (Notch3) across the various differentiated states of thyroid cancer and determine its clinical impact. METHODS Notch3 expression was analyzed in a tissue microarray of normal and pathologic thyroid biopsies from 155 patients. The functional role of Notch3 was then investigated by upregulating its expression in a follicular thyroid cancer (FTC) cell line. RESULTS Notch3 expression regressed across decreasingly differentiated, increasingly malignant thyroid specimens, correlated with clinicopathological attributes reflecting poor prognosis, and independently predicted survival following univariate and multivariate analysis. Overexpression of the active Notch3 intracellular domain (NICD3) in an FTC, gain-of-function line led to functional CBF1-binding and increased thyroid-specific gene transcription. NICD3 induction also reduced tumor burden in vivo, and initiated the intrinsic apoptotic cascade, alongside suppressing cyclin and Bcl-2 family expression. CONCLUSIONS Loss of Notch3 expression may be fundamental to the process of dedifferentiation that accompanies thyroid oncogenesis. Conversely, activation of Notch3 in thyroid cancer exerts an antiproliferative effect and restores elements of a differentiated phenotype. These findings provide preclinical rationale for evaluating Notch3 as a disease prognosticator and therapeutic target in advanced thyroid cancer.