Kate I. Patterson

Dual-specificity phosphatases: critical regulators with diverse cellular targets

DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. Of these subgroups, a great deal of research has focused on the characterization of the MKPs. As their name suggests, MKPs dephosphorylate MAPK (mitogen-activated protein kinase) proteins ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 with specificity distinct from that of individual MKP proteins. Atypical DUSPs are mostly of low-molecular-mass and lack the N-terminal CH2 (Cdc25 homology 2) domain common to MKPs. The discovery of most atypical DUSPs has occurred in the last 6 years, which has initiated a large amount of interest in their role and regulation. In the past, atypical DUSPs have generally been grouped together with the MKPs and characterized for their role in MAPK signalling cascades. Indeed, some have been shown to dephosphorylate MAPKs. The current literature hints at the potential of the atypical DUSPs as important signalling regulators, but is crowded with conflicting reports. The present review provides an overview of the DUSP family before focusing on atypical DUSPs, emerging as a group of proteins with vastly diverse substrate specificity and function.

Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer

Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.

Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer

Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Integrative genome-wide expression and promoter DNA methylation profiling identifies a potential novel panel of ovarian cancer epigenetic biomarkers

To identify epigenetic-based biomarkers for diagnosis of ovarian cancer we performed MeDIP-Chip in A2780 and CaOV3 ovarian cancer cell lines. Validation by Sequenom massARRAY methylation analysis confirmed a panel of six gene promoters (ARMCX1, ICAM4, LOC134466, PEG3, PYCARD & SGNE1) where hypermethylation discriminated 27 serous ovarian cancer clinical samples versus 12 normal ovarian surface epithelial cells (OSE) (ROC of 0.98). Notably, CpG sites across the transcription start site of a potential long-intergenic non-coding RNA (lincRNA) gene (LOC134466), was shown to be hypermethylated in 81% of serous EOC and could differentiate tumours from OSE (p<0.05). We propose that this potential biomarker panel holds great promise as a diagnostic test for high-grade (Type II) serous ovarian cancer.

Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential. Cancer Epidemiol Biomarkers Prev; 20(1); 148–59. ©2011 AACR.

Expression of urokinase plasminogen activator and its receptor in advanced epithelial ovarian cancer patients

BACKGROUND The urokinase plasminogen activator (uPA) system has been implicated in progression and poor prognosis in epithelial ovarian cancer (EOC) patients. The present study investigated the distribution of uPA and its receptor (uPAR) in EOC cell lines, primary and metastatic tumors, and the relationship between uPA/uPAR and matrix metalloproteinase (MMP) expression using immunohistochemistry. We also studied the association between uPA/uPAR expression and clinical and pathological parameters including disease progression free survival (PFS). METHODS The expression of uPA/uPAR was examined on paraffin-embedded tissue sections from primary EOC (n=100), and matched metastatic lesions (n=30) of untreated patients, normal ovarian tissues (n=20) as well as 8 primary and metastatic EOC cell lines by immunohistochemistry. Co-immunolabeling of uPA and MMP-1, -2, -9 or MT1-MMP was examined using confocal microscopy. RESULTS The expression of uPA/uPAR was found in most primary (92% and 88% positive, respectively), metastatic ovarian tumors (93% and 90% positive, respectively), and all of examined EOC cell lines. The majority of specimens showed moderate to strong immunostaining of tumor and stromal cells; for primary specimens, this was significantly associated with tumor stage, grade and time to relapse (P<0.01). Overexpression of uPA/uPAR was found to be associated with an unfavorable prognosis with significantly reduced median disease PFS of 16 vs. 33 months for uPA (P<0.001), and 15 vs. 28 months for uPAR (P<0.001). Co-localization of uPA with MMP-1, -2, -9 or MT1-MMP was also seen in primary tumors and metastatic lesions. CONCLUSIONS The expression of uPA/uPAR was associated with EOC progression. uPA/uPAR are useful markers for EOC prognosis and could be promising therapeutic targets for treating incurable, recurrent EOC.

Monoclonal antibody targeting MUC1 and increasing sensitivity to docetaxel as a novel strategy in treating human epithelial ovarian cancer

The purpose of this study was to investigate the in vitro effect of anti-MUC1 monoclonal antibody (MAb) C595 alone and in combination with docetaxel, on the growth and survival of different epithelial ovarian cancer (EOC) cell lines. MUC1 expression was assessed on EOC cell lines (OVCAR-3, IGROV-1, A2780, CAOV-3, TOV-21G, TOV-112D, SKOV-3 and OV-90) using immunofluorescence labeling and flow cytometry. The effect of MAb C595 alone or in combination with docetaxel on the cell lines was studied by proliferation, colony and TUNEL assays. Our results indicate that all primary and metastatic EOC cell lines tested were positive to MAb C595 (MUC1); MAb C595 inhibited EOC cell proliferation in a MUC1- and dose-dependent manner; low-dose MAb C595 (1/2 of IC₅₀) combined with docetaxel greatly improved efficiency of cell killing in EOC cells and induced apoptosis; the additive effect of MAb C595 was further confirmed in colony forming assays; and cell death following single or combined treatments was associated with the release of cytochrome c and increased caspase-3 activity. These results suggest that MAb C595 used either alone, or combined with docetaxel, is an attractive strategy for targeting human EOC.

DUSP26 negatively affects the proliferation of epithelial cells, an effect not mediated by dephosphorylation of MAPKs

Dual specificity phosphatases are characterised by their ability to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues within the one substrate. The aim of this study was to characterise the phosphatase activity of the atypical dual specificity phosphatase, DUSP26 on MAP kinases, and to determine its expression, regulation and function in cancer cells. Overexpression and knockdown of DUSP26 in epithelial cells and in vitro phosphatase assays were used to demonstrate that, contrary to several published reports, DUSP26 does not act as a dual specificity phosphatase on ERK, JNK or p38 MAPKs. However, overexpression of DUSP26 in MCF10A epithelial cells suppressed colony formation and acinar growth in 3D culture, effects dependent on its phosphatase activity, while knockdown of DUSP26 in HOSE17.1 cells enhanced colony formation and cellular proliferation. DUSP26 mRNA expression was reduced in neuroblastoma, brain and ovarian cancer cell lines. Consistent with epigenetic silencing of DUSP26, expression was enhanced by treatment of cells with 5-aza-2-deoxycitidine and trichostatin A, and a CpG island upstream of the DUSP26 transcriptional start site was variably methylated in cancer cell lines. Together, these results help to clarify confusion in the literature relating to DUSP26 substrate specificity and support recent reports that substrates other than MAPKs are the primary substrates of this phosphatase. In addition, they indicate that DUSP26 may function as a tumour suppressor in particular cancers.

Germinal center antibody mutation trajectories are determined by rapid self/foreign discrimination

Autoantibody redemption through rapid mutations Antibodies distinguish foreign epitopes from closely related self-antigens by poorly understood mechanisms. In mice, Burnett et al. found that a proportion of B cells could cross-react with similar foreign and self-antigens (see the Perspective by Kara and Nussenzweig). Challenge with self-antigen resulted in anergy (i.e., a lack of immune response), which was reversed by exposure to high-density foreign antigen. Mutations that decreased self-affinity were rapidly selected for, whereas selection for epistatic mutations that enhanced foreign reactivity took longer. Self-reactivity, rather than being an impediment to immunization, resulted in higher affinities against a foreign immunogen. Science, this issue p. 223; see also p. 152 The rapid mutations of autoantibodies target foreign-antigen look-alikes. Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.