Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

Biochemical Characterization of Yeast Xrn1.

Messenger RNA degradation is an important component of overall gene expression. During the final step of eukaryotic mRNA degradation, exoribonuclease 1 (Xrn1) carries out 5' → 3' processive, hydrolytic degradation of RNA molecules using divalent metal ion catalysis. To initiate studies of the 5' → 3' RNA decay machinery in our lab, we expressed a C-terminally truncated version of Saccharomyces cerevisiae Xrn1 and explored its enzymology using a second-generation, time-resolved fluorescence RNA degradation assay. Using this system, we quantitatively explored Xrn1's preference for 5'-monophosphorylated RNA substrates, its pH dependence, and the importance of active site mutations in the molecule's conserved catalytic core. Furthermore, we explore Xrn1's preference for RNAs containing a 5' single-stranded region both in an intermolecular hairpin structure and in an RNA-DNA hybrid duplex system. These results both expand and solidify our understanding of Xrn1, a centrally important enzyme whose biochemical properties have implications in numerous RNA degradation and processing pathways.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-ß expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Douching, Talc Use, and Risk for Ovarian Cancer and Conditions Related to Genital Tract Inflammation.

BACKGROUND: Douching is associated with disorders involving genital tract inflammation and genital talc use with epithelial ovarian cancer (EOC), but their joint effects are infrequently considered. METHODS: From 2,040 cases of EOC and 2,100 controls enrolled in eastern Massachusetts and New Hampshire, we used unconditional logistic regression to estimate risk for EOC associated with douching and/or talc use. In subsets of cases and controls, we also collected information about pelvic inflammatory disease (PID), ectopic pregnancy, and cervical neoplasia to estimate risk for these events from douching and/or talc use. RESULTS: The adjusted OR and 95% confidence interval (CI) for all EOC was 0.94 (0.76-1.16) in women who douched but never used talc and 1.28 (1.09-1.51) in women who used talc but never douched. Compared with women who never regularly douched or used talc, ORs (95% CIs) were 0.83 (0.52-1.33) for women who both used talc and homemade douches and 1.53 (1.11-2.10) for women who both used talc and store-bought douches. Cases who both douched and used talc were more likely to have had PID compared with cases who had used neither [OR = 5.03 (95% CI, 1.61-15.7)]. CONCLUSIONS: Douching is not an independent risk factor for ovarian cancer, but the combination of talc use and store-bought douches may modestly increase the risk for EOC beyond that for talc use alone. IMPACT: The joint effect of talc use and douching, especially with commercial products, should be considered in evaluating risks associated with disorders involving genital tract inflammation or EOC.

Migration of Talc From the Perineum to Multiple Pelvic Organ Sites.

OBJECTIVES: Genital talc use is associated with increased risk for ovarian carcinoma in epidemiologic studies. Finding talc in pelvic tissues in women with ovarian carcinoma who have used talc is important in documenting exposure and assessing talc's biologic potential, but tissue-based morphology studies have been rarely reported. METHODS: We report five patient cases with documented perineal talc use, each of whom had talc (by both polarized light and scanning electron microscopy) in multiple pelvic sites distant from the perineum. Six negative-exposure control patients were also analyzed. RESULTS: Talc particles were found in exposed patients, typically within two or more of the following locations: pelvic region lymph nodes, cervix, uterine corpus, fallopian tubes, and ovaries. CONCLUSIONS: Our report adds new insights into the biologic potential of talc and suggests additional anatomic sites that should be closely examined for talc by oncologic surgical pathologists in the setting of perineal talc use.

Correlative polarizing light and scanning electron microscopy for the assessment of talc in pelvic region lymph nodes.

Perineal talc use is associated with ovarian carcinoma in many case-control studies. Such talc may migrate to pelvic organs and regional lymph nodes, with both clinical and legal significance. Our goal was to differentiate talc in pelvic lymph nodes due to exposure, versus contamination with talc in the laboratory. We studied 22 lymph nodes from ovarian tumor patients, some of which had documented talc exposure, to quantify talc using digestion of tissue taken from paraffin blocks and scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX). Talc particles correlated significantly with surface contamination assessments using polarized light microscopy. After adjusting for surface contamination, talc burdens in nodes correlated strongly with perineal talc use. In a separate group of lymph nodes, birefringent particles within the same plane of focus as the tissues in histological sections were highly correlated with talc particles within the tissue by in situ SEM/EDX (r = 0.80; p < 0.0001). We conclude that since talc can be a surface contaminant from tissue collection/preparation, digestion measurements may be influenced by contamination. Instead, because they preserve anatomic landmarks and permit identification of particles in cells and tissues, polarized light microscopy and in situ SEM/EDX are recommended to assess talc in lymph nodes.

Assessment of a Chemotherapy Response Score (CRS) System for Tubo-Ovarian High-Grade Serous Carcinoma (HGSC).

A chemotherapy response score (CRS) system was recently described to assess the histopathologic response and prognosis of patients with tubo-ovarian high-grade serous carcinoma (HGSC) receiving neoadjuvant chemotherapy. The current study was performed as an independent assessment of this CRS system. We retrospectively identified advanced stage HGSC patients who received neoadjuvant chemotherapy and underwent interval debulking. If available, a hemotoxylin and eosin slide from the omentum and the adnexa was selected for the study. Slides were independently scored by 13 pathologists using the 3-tiered CRS system. Reviewers then received web-based training and rescored the slides. Overall survival and progression-free survival were estimated using the Kaplan-Meier method and compared using the log-rank test. A total of 68 patients with omental (n=65) and/or adnexal (n=59) slides were included in the study. Interobserver reproducibility was moderate for omentum (κ, 0.48) and poor for adnexa (κ, 0.40), which improved for omentum (κ, 0.62) but not for adnexa (κ, 0.38) after online training. For omental slides, a consensus CRS of 1/2 was associated with a shorter median progression-free survival (10.9 mo; 95% confidence interval, 9-14) than a CRS of 3 (18.9 mo; 95% CI, 18-24; P=0.020). In summary, a 3-tiered CRS system of hemotoxylin and eosin-stained omental deposits can yield prognostic information for HGSC patients receiving neoadjuvant chemotherapy, and web-based training improved reproducibility but did not alter determination of clinical outcomes. The CRS system may allow oncologists to identify potential nonresponders and triage HGSC patients for heightened observation and/or clinical trials.

Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube.

BACKGROUND: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors. METHODS: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors. RESULTS: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFß1 and TGFß2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection. CONCLUSIONS: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

Cyclin A1 expression and paclitaxel resistance in human ovarian cancer cells.

BACKGROUND: The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. METHODS: Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by ß-galactosidase activity staining. RESULTS: Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated ß-galactosidase activity. CONCLUSIONS: Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.