Improving screening and treatment options for patients with epithelial ovarian cancer has been a major challenge in cancer research. Development of novel diagnostic and therapeutic approaches, particularly for the most common subtype, high-grade serous ovarian cancer (HGSC), has been hampered by controversies over the origin of the disease and a lack of spontaneous HGSC models to resolve this controversy. Over long-term culture in our laboratory, an ovarian surface epithelial (OSE) cell line spontaneously transformed OSE (STOSE). The objective of this study was to determine if the STOSE cell line is a good model of HGSC. STOSE cells grow faster than early passage parental M0505 cells with a doubling time of 13 and 48 h, respectively. STOSE cells form colonies in soft agar, an activity for which M0505 cells have negligible capacity. Microarray analysis identified 1755 down-regulated genes and 1203 up-regulated genes in STOSE compared to M0505 cells, many associated with aberrant Wnt/β-catenin and Nf-κB signaling. Upregulation of Ccnd1 and loss of Cdkn2a in STOSE tumors is consistent with changes identified in human ovarian cancers by The Cancer Genome Atlas. Intraperitoneal injection of STOSE cells into severe combined immunodeficient and syngeneic FVB/N mice produced cytokeratin+, WT1+, inhibin−, and PAX8+ tumors, a histotype resembling human HGSC. Based on evidence that a SCA1+ stem cell-like population exists in M0505 cells, we examined a subpopulation of SCA1+ cells that is present in STOSE cells. Compared to SCA1− cells, SCA1+ STOSE cells have increased colony-forming capacity and form palpable tumors 8 days faster after intrabursal injection into FVB/N mice. This study has identified the STOSE cells as the first spontaneous murine model of HGSC and provides evidence for the OSE as a possible origin of HGSC. Furthermore, this model provides a novel opportunity to study how normal stem-like OSE cells may transform into tumor-initiating cells.

Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquitin ligase or ligases that recognize ligand bound PPARγ is an essential step in determining the physiological significance of the relationship between activation and ubiquitin-dependent degradation of PPARγ. Using an RNA interference-based screen, we identified five RING (really interesting new gene)-type ubiquitin ligases that alter PPARγ protein levels in adipocytes. Here, we demonstrate that Drosophila seven-in-absentia homolog 2 (Siah2), a mammalian homolog of Drosophila seven-in-absentia, regulates PPARγ ubiquitylation and ligand-dependent activation of PPARγ in adipocytes. We also demonstrate that Siah2 expression is up-regulated during adipogenesis and that PPARγ interacts with Siah2 during adipogenesis. In addition, Siah2 is required for adipogenesis. These data suggest that modulation of PPARγ protein levels by the ubiquitin ligase Siah2 is essential in determining the physiological effects of PPARγ activation in adipocytes.

BackgroundBone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches.ResultsWe developed a simple approach to isolate bone RNA that combines pulverizing the bone and the phenol-guanidinium based RNA extraction in a single step while maintaining near-freezing temperatures. This single step method increases the yield of high quality RNA by eight-fold, with RNA integrity numbers ranging from 6.7 to 9.2.ConclusionsOur streamlined approach substantially increases the yield of high-quality RNA from bone tissue while facilitating safe and efficient processing of multiple samples using readily available platforms. The RNA obtained from this method is suitable for use in gene expression analysis in real-time quantitative PCR, microarray, and next generation sequencing applications.

Objective Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, we examined the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation. Methods Wild-type and Siah2KO mice were fed a low or high fat diet for 16 weeks. Indirect calorimetry, body composition, glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution and lipolysis were also analyzed. Results Enlarged adipocytes in obese Siah2KO mice are not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis and crown-like structures are reduced in the Siah2KO adipose tissue and Siah2KO adipocytes are more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increases expression of PPARγ target genes involved in lipid metabolism and decreases expression of proinflammatory adipokines regulated by PPARγ. Conclusions Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation.