Cheung Wong

Factor V Leiden, prothrombin 20210A and the risk of venous thrombosis among cancer patients

Cancer patients have an increased risk of venous thrombosis (VT). The association of factor V Leiden (FVL) and the prothrombin 20210A variant with VT in cancer patients is not established. We genotyped 101 cancer patients with VT and 101 cancer patients without VT for these polymorphisms. Five cases and three controls were heterozygous for FVL, yielding an odds ratio of 1·7 (95% confidence interval (CI) 0·3–10·7). Five cases and no controls were heterozygous for prothrombin 20210A, for an odds ratio of 6·7 (95% CI 0·9–infinity). Prothrombin 20210A may be associated with VT risk among cancer patients.

NEGATIVE REGULATION OF HIF-1α BY AN FBW7-MEDIATED DEGRADATION PATHWAY DURING HYPOXIA

Hypoxia inducible factor‐1α (HIF‐1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF‐1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel–Lindau; however, little is known about the regulation of HIF‐1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3β (GSK3β) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF‐1α phosphorylation by GSK3β in hypoxia leads to interaction with FBW7 and ubiquitin‐dependent degradation. Expression of constitutively active GSK3β reduced HIF‐1α protein and transcriptional activity and increased ubiquitination of HIF‐1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3β promoted HIF‐1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF‐1α stabilization when FBW7 was overexpressed and both the elevation of HIF‐1α levels and decrease in ubiquitinated HIF‐1α when FBW7 was suppressed. Furthermore, HIF‐1α associated with FBW7γ by co‐immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3β phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK‐OV‐3 cell lines expressing suppressed GSK3β or FBW7. These data introduce a new mechanism for regulation of HIF‐1α during hypoxia that utilizes phosphorylation to target HIF‐1α for ubiquitin‐dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis. J. Cell. Biochem. 112: 3882–3890, 2011.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

BackgroundOvarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression.MethodsProtein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α).ResultsThere was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062).ConclusionsTARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.

Aspiration Cytology of Ovarian Cystic Masses: Histologic Correlation and Review of the Literature

Objective: To determine the diagnostic accuracy of cytologic evaluation of ovarian cystic masses. Study Design: Sixty-seven ovarian cystic masses with fine needle aspiration cytology and concurrent or subsequent cystectomy/oophorectomy with histology were examined. Correlations with malignancy were made with 4 parameters: serum CA-125, radiographic size and architecture, and cytology. Results: Histologic examination of the 67 cases revealed 10 malignancies including 3 primary ovarian carcinomas, 2 metastatic neoplasms, and 5 borderline tumors. In the 10 malignant cases, the cytologic diagnoses were that of benign (n = 2), benign but non-diagnostic/paucicellular (n = 3), and atypical/malignant (n = 5), giving an overall sensitivity for cytology of 50%. However, there were no false positives (specificity of 100%). Reasons for the low sensitivity of cytology were the paucicellular nature of aspirate (n = 3), focality of ovarian borderline tumors (n = 5), and surface involvement by metastatic cancer (n = 2). The 4 parameters were independent of one another and none proved to have significant correlation with malignancy (p > 0.05). Thirty-nine percent of the aspirates had low cellularity (6% non-diagnostic/33% pauci-cellular). Conclusions: Cytology was the only parameter with 100% specificity and 100% positive predictive value. However, paucicellular specimens are a common problem in aspiration from this site.

Recurrent pregnancy loss in a woman with NLRP7 mutation: not all molar pregnancies can be easily classified as either "partial" or "complete" hydatidiform moles.

Recurrent hydatidiform moles is an uncommon occurrence. Over the past decade, genetic studies of women with multiple recurrent molar pregnancies have revealed that maternal mutations in two different genes, NLRP7 and C6orf221, result in recurrent moles. We report a 23 year old woman, born of unrelated parents, who has experienced three molar pregnancies in succession. Whilst the first pregnancy was classified as a complete hydatidiform mole, the second and third moles defied classification as complete or partial mole using conventional histology, p57 nuclear staining pattern and ploidy studies. Molecular and cytogenetic studies proved that all three molar pregnancies were diploid and biparental in origin. Gene sequencing analysis showed that the patient is homozygous for a previously described mutation in NLRP7. A SNP microarray ruled out the presence of deletion of the NLRP7 locus. This case draws attention to the fact that recurrent molar pregnancies may be the result of specific, identifiable gene mutations, even in patients from non-consanguineous backgrounds. When pathologists encounter patients with molar pregnancies that are diploid and p57 negative and yet have fetal elements such as nucleated red blood cells or immature fetal tissues, it should heighten their suspicion of a possible genetic basis and appropriate molecular genetic workup performed with counseling offered.

Abstract POSTER-BIOL-1335: Threonyl-tRNA synthetase overexpression correlates with angiogenesis and progression of human ovarian cancer

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. There is growing evidence that tRNA synthetases have a role in cell signaling separate from their function in protein synthesis. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity. Exploration of protein and mRNA databases further showed a connection between TARS expression and ovarian cancer. The objective of this study was to test the hypothesis that TARS is secreted by ovarian cancer cells and that its expression in clinical samples correlates with angiogenic markers and ovarian cancer progression. TARS secretion by SKOV3 human ovarian cancer cells was induced by hypoxia or tumor necrosis factor-α (TNF-α) as detected by ELISA and Western blot of culture media (p<0.001). For the clinical study, TARS, vascular endothelial growth factor (VEGF) and PECAM were assessed by immunohistochemistry in serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA125, and tumor necrosis factor-α (TNF-α). There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p<0.001). TARS expression and localization were also correlated with VEGF (p<0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p=0.017). Patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p=0.062). Together these results show that TARS is secreted in response to cell stress and that its expression is increased in epithelial ovarian cancer according to stage and angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer. Citation Format: Theresa L. Wellman, Midori Eckenstein, Cheung Wong, Mercedes Rincon, Takamaru Ashikaga, Sharon L. Mount, Christopher S. Francklyn, Karen M. Lounsbury. Threonyl-tRNA synthetase overexpression correlates with angiogenesis and progression of human ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1335.

Abstract 3092: FBW7 targets hypoxia inducible factor-1α (HIF-1α) for proteasomal degradation during hypoxia

Hypoxia Inducible Factor-1α (HIF-1α) mediates expression of genes associated with endothelial cell-mediated angiogenesis and is associated with poor outcomes in a variety of cancers. In normoxia, HIF-1α is ubiquitinated and degraded through interactions with the E3 ubiqutin ligase, von Hippel-Lindau (vHL); however, little is known about the negative regulation of HIF-1α in hypoxia. FBW7, an E3 ubiquitin ligase, has been shown to interact with several transcription factors including those phosphorylated by glycogen synthase kinase 3β (GSK3β). The current study tested the hypothesis that phosphorylation of HIF-1α by GSK3β increases the FBW7-mediated ubiquitination and degradation of HIF-1α, thereby resulting in suppression of the hypoxia-mediated angiogenic response in SKOV-3 ovarian cancer cells. HIF-1α protein and VEGF transcript levels in hypoxia were increased when GSK3β activity was inhibited and were reduced by expression of constitutively active GSK3β (GSK3S9A). Additionally, expression of GSK3S9A increased HIF-1α ubiquitination. Conditioned media from SKOV-3 cells expressing shRNA against GSK3β (shGSK3) had an enhanced effect on endothelial tube formation in a Matrigel matrix, suggesting that GSK3β exerts an inhibitory effect on hypoxia-mediated angiogenesis. Overexpression of the β and γ isoforms of FBW7 decreased HIF-1α stabilization, and HIF-1α interacts with FBW7 by co-immunoprecipitation. Furthermore, knockdown of FBW7 using siRNA resulted in increased HIF-1α levels in hypoxia. These data suggest a new mechanism for negative regulation of HIF-1α during hypoxia that utilizes phosphorylation by GSK3β and interaction with FBW7 leading to ubiquitination and proteasomal degradation. Results of this study better define the signaling pathways necessary for HIF-1α-mediated signaling and may identify new targets that mediate angiogenesis in disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2011-3092