Michael A Hahn

Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer

BackgroundThere is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers.MethodsTo identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification.ResultsmicroRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784).ConclusionsWe identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

The chemokine CXCL1 induces proliferation in epithelial ovarian cancer cells by transactivation of the epidermal growth factor receptor

The chemokine CXCL1 is elevated in plasma and ascites from patients with ovarian cancer. We have previously shown that CXCL1 is a marker of phosphatidylinositol 3-kinase signalling in epithelial ovarian cancer (EOC) cell lines, a pathway that is commonly activated in ovarian tumours. To investigate whether CXCL1 also has functional significance in ovarian cancer, this chemokine was either down-regulated using siRNAs or overexpressed by transfection of CXCL1 into the EOC cell lines SKOV3 and OVCAR-3 and proliferation assessed over 7 days. Overexpression of CXCL1 increased proliferation of ovarian cancer cells over 7 days, while down-regulation was inhibitory. Treatment of cells with recombinant CXCL1 induced epidermal growth factor receptor (EGFR) phosphorylation at Y1068, indicating crosstalk between the CXCL1 G-protein-coupled receptor CXCR2 and the EGFR. CXCL1-induced proliferation was also decreased by inhibition of EGFR kinase activity and was dependent on extracellular matrix metalloproteinase-mediated release of heparin-binding EGF (HB-EGF). Involvement of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signalling was also evident since inhibition of both Ras and MEK activity decreased CXCL1-induced proliferation. CXCL1-induced ERK1/2 phosphorylation was inhibited by the MEK1 inhibitor PD98059; however, EGFR phosphorylation was unaffected, indicating that CXCL1 activation of MAPK signalling is downstream of the EGFR. Taken together, these data show that CXCL1 functions through CXCR2 to transactivate the EGFR by proteolytic cleavage of HB-EGF, leading to activation of MAPK signalling and increased proliferation of EOC cells.

The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination

Monoubiquitination of histone H2B is a dynamic post-translational histone modification associated with transcriptional elongation and the DNA damage response. To date, dysregulation of histone monoubiquitination has not been linked to pathogenic mutations in genes encoding proteins, or co-factors, catalyzing this modification. The tumor suppressor cell division cycle 73 (CDC73) is mutated and/or down-regulated in parathyroid carcinoma, renal, breast, gastric and colorectal tumors, as well as in the germline of patients with the familial disorder-hyperparathyroidism jaw tumor syndrome. Using CDC73 as bait in a yeast two-hybrid assay, we identified the ring finger proteins RNF20 and RNF40 as binding partners of this tumor suppressor. These polypeptides constitute a heterodimeric complex that functions as the E3 ubiquitin ligase for monoubiquitination of histone H2B at lysine 120 (H2B-K120). We show that RNF20 and RNF40 bind to discrete, but closely located, residues on CDC73. Monoubiquitinated H2B-K120 was significantly reduced after loss of nuclear CDC73, both in vitro upon down-regulation of CDC73, and in CDC73 mutant parathyroid tumors. A second histone modification, trimethylation of histone 3 at lysine 4 (H3-K4me3), remained unchanged in the presence of mutant or down-regulated CDC73, suggesting that H3-K4me3 is not always tightly linked to H2B-K120 monoubiquitination for transcription as previously described. This is the first report of pathogenic mutations affecting histone monoubiquitination. We conclude that CDC73 is required for the maintenance of H2B-K120 monoubiquitination and propose that reduction in levels of monoubiquitinated H2B-K120 is a major mechanism whereby mutations in CDC73 exert their tumorigenic effect.

Identification of a functional bipartite nuclear localization signal in the tumor suppressor parafibromin

Parafibromin is a putative tumor suppressor encoded by HRPT2, mutations in which have been implicated in the familial tumor syndrome hyperparathyroidism jaw tumor syndrome (HPT-JT), and sporadic parathyroid carcinoma. Recently, parafibromin has been shown to be an accessory factor for RNA polymerase II as part of the human Paf1 complex, suggesting, as has been shown for its yeast homologue (Cdc73), that it may have a role as an important regulator of transcription. Parafibromin has also been shown to interact with a histone methyltransferase complex that methylates histone H3 and to inhibit proliferation when overexpressed in mammalian cell lines. Despite these findings, the cellular localization of parafibromin has been controversial, with reports of both nuclear and nucleocytoplasmic localization. We have expressed wild-type and mutant parafibromin tagged with enhanced green fluorescent protein and have identified a functional bipartite nuclear localization signal (NLS) at residues 125–139 (nucleotides 373–417), KRAADEVLAEAKKPR, that is evolutionarily conserved and critical for the nuclear localization of parafibromin. We have also shown that the C-terminal arm of this bipartite NLS plays the primary role in nuclear localization. In support of these findings, specific HRPT2 mutations identified in HPT-JT or sporadic parathyroid carcinoma predicted to truncate parafibromin upstream of or within this NLS disrupt nuclear localization.

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125–139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76–92, 192–194 and 393–409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromins bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.

Molecular diagnosis of primary hyperparathyroidism in familial cancer syndromes

In the last few years, causative genes have been identified for most of the familial hyperparathyroidism conditions. Germline mutations in the tumour suppressors multiple endocrine neoplasia type 1 (MEN1) and hyperparathyroidism 2 (HRPT2) provide a molecular diagnosis of multiple endocrine neoplasia type 1 and hyperparathyroidism jaw tumour syndrome, respectively. Germline mutations in the proto-oncogene RET (rearranged during transfection) provide a molecular diagnosis of multiple endocrine neoplasia type 2. Germline mutations of both MEN1 and, less frequently HRPT2, have been found in familial isolated hyperparathyroidism. A molecular diagnosis can now be incorporated into the management of patients with these conditions, however, the ease of diagnostics and value of genetic information in the context of clinical screening and early surgical intervention varies between these disorders. This review focuses on familial hyperparathyroidism and its known causative genes in the setting of neoplastic syndromes, with particular discussion of recent developments in the molecular diagnosis of parathyroid carcinoma.

Genomic alterations as mediators of miRNA dysregulation in ovarian cancer

Ovarian cancer is the fifth most common cause of cancer death in women worldwide. Serous epithelial ovarian cancer (SEOC) is the most common and aggressive histological subtype. Widespread genomic alterations go hand‐in‐hand with aberrant DNA damage signaling and are a hallmark of high‐grade SEOC. MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that are nonrandomly distributed in the genome. They are frequently located in chromosomal regions susceptible to copy number variation (CNV) associated with malignancy that can influence their expression. Widespread changes in miRNA expression have been reported in multiple cancer types including ovarian cancer. This review examines CNV and single nucleotide polymorphisms, two common types of genomic alterations that occur in ovarian cancer, in the context of their influence on the expression of miRNA and the ability of miRNA to bind to and regulate their target genes. This includes genes encoding proteins involved in DNA repair and the maintenance of genomic stability. Improved understanding of mechanisms of miRNA dysregulation and the role of miRNA in ovarian cancer will provide further insight into the pathogenesis and treatment of this disease.

The effect of disease-associated HRPT2 mutations on splicing

Mutations in the tumour suppressor HRPT2 occur in patients with parathyroid carcinoma, kidney tumours and Hyperparathyroidism-Jaw Tumour syndrome. Disruption of exonic splicing through mutation of donor/acceptor splice sites or exonic splice enhancer (ESE) sites leads to loss of function of a number of major tumour suppressors including BRCA1, APC and MLH1. Given that the effect of HRPT2 mutations on splicing has not been widely studied, we used an in vitro splicing assay to determine whether 17 HRPT2 mutations located in hot-spot and other exons predicted to disrupt ESE consensus sites led to aberrant splicing. Using two independent web-based prediction programs, the majority of these mutations were predicted to disrupt ESE consensus sites; however, aberrant splicing of HRPT2 transcripts was not observed. Canonical donor or acceptor splice site mutations were also investigated using this splicing assay and transcripts assessed from tumour tissue. Splice site mutations were shown to lead to either exon skipping or retention of intronic sequences through the use of cryptic splice sites comprised of non-classical splicing signals. Aberrant splicing caused by disruption of ESE sites does not appear to have a major role in HRPT2-associated disease; however, premature truncation of parafibromin as the result of canonical donor or acceptor splice site mutations is associated with pathogenicity. Functional splicing assays must be undertaken in order to confirm web-based software predictions of the modification of putative ESE sites by disease-associated mutations.

Abstract 3150: miR-100 in ovarian cancer cell lines

MicroRNA (miRNA), small non-coding RNA sequences that regulate gene expression, have the potential to function as tumor suppressors or oncogenes. To determine the contribution of miRNA to ovarian oncogenesis this project sought to identify dysregulated miRNA and their targets in ovarian cancer cell lines. miRNA expression profiling was performed on 6 serous epithelial ovarian cancer (SEOC) cell lines and normal ovarian surface epithelial (OSE) cells. miRNAs differentially expressed between SEOC and OSEs were selected for further analyses and candidate target genes identified by prediction algorithms. miRNA expression was restored by transient transfection of miRNA mimics or stable transduction of lentiviral vectors expressing miRNA precursors. The effects on the expression of 2 predicted targets, SMARCA5 and FRAP1, were investigated using a luciferase reporter system, quantitative RT-PCR (qPCR) and immunoblotting. The effect of miRs on cellular proliferation was also explored. Expression profiling identified miR-100 as having >5-fold lower expression in SEOC vs OSE cells. Seed sequences or binding sites for miR-100 were identified in the 3′UTRs of SMARCA5 and FRAP1. Using a luciferase expression system in OVCAR-3 cells, a miR-100 mimic repressed expression of SMARCA5 3′UTR by 40 % (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3150. doi:1538-7445.AM2012-3150

Abstract 2167: The tumor suppressor CDC73/parafibromin is required for the maintenance of histone 2B monoubiquitination both in vitro and in vivo

CDC73 (cell division cycle 73), also known as parafibromin, is a ubiquitously expressed tumor suppressor that is mutated in the germline of patients with the familial disorder Hyperparathyroidism Jaw Tumor syndrome, as well as somatically mutated in sporadic parathyroid cancer. Furthermore, CDC73 has been implicated in breast, gastric, renal and colorectal tumorigenesis. CDC73 is a member of the PAF1 (RNA polymerase II-associated factor 1) transcriptional complex (PAF1c), along with PAF1, CTR9, LEO1, and SKI8. Consistent with roles in transcription, CDC73 is localized to the nucleus, and has also been reported in the nucleolus. This tumor suppressor has been shown to function as a negative regulator of cell cycle progression and to promote apoptosis, as well as have a role in the regulation of 3′ processing of histone mRNA. We used CDC73 as bait in a yeast two-hybrid assay and identified the E3 ubiquitin ligase ring finger proteins RNF20 and RNF40 as CDC73 binding partners. RNF20 and RNF40 exist in a heterodimeric complex that functions to monoubiquitinate histone H2B at lysine 120 (H2B-K120). Monoubiquitination of H2B-K120 is associated with transcription in undamaged cells, is induced after DNA damage, and has a role in the maintenance of replication-dependent histone mRNA 3′ - end processing. We have confirmed the interaction of CDC73 with both RNF20 and RNF40 in mammalian cells by co-immunoprecipitation of overexpressed and endogenous proteins from human embryonic kidney (HEK293) cells. Down-regulation of endogenous CDC73 by siRNA in HEK293 cells and the breast cancer cell line MCF7 led to a significant reduction of monoubiquitinated H2B-K120. Furthermore, in a series of eleven parathyroid carcinomas harboring mutations in CDC73 that led to loss of nuclear CDC73, monoubiquitinated H2B-K120 was either absent or significantly reduced relative to benign parathyroid tumors that expressed nuclear CDC73. siRNA down-regulation of other PAF1c members in MCF7 cells also led to reduction in levels of monoubiquitinated H2B-K120. Given that CDC73 is the only PAF1c member in which cancer-associated mutations have been reported, the translational significance of these findings are unclear. Loss of the CTR9 locus has been reported in pancreatic cancer, as well as overexpression of PAF1. PAF1c members may have a greater role in malignancy than previously described. This is the first report to demonstrate pathogenic mutations affecting monoubiquitination of a histone. We propose that loss of H2B-K120 monoubiquitination is a major mechanism whereby CDC73 mutations exert their tumorigenic effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2167. doi:1538-7445.AM2012-2167