Lysine-specific demethylase KDM3A regulates ovarian cancer stemness and chemoresistance.

This corrects the article DOI: 10.1038/onc.2016.320.

Lysine-specific demethylase KDM3A regulates ovarian cancer stemness and chemoresistance.

Ovarian cancer is the leading cause of death among all gynecological malignancies due to the development of acquired chemoresistance and disease relapse. Although the role of cancer stem cells (CSCs), a subset of tumor cells with the self-renewal and differentiation capabilities, in therapeutic resistance is beginning to be better understood, the significance of epigenetic regulatory mechanisms responsible for integrating the stemness with drug resistance remain poorly understood. Here we identified that lysine demethylase KDM3A as a critical regulator of ovarian cancer stemness and cisplatin resistance by inducing the expressions of pluripotent molecules Sox2 and Nanog and anti-apoptotic B-cell lymphoma 2 (Bcl-2), respectively. In addition, KDM3A induces ovarian cancer growth while antagonizing cellular senescence by repressing the expression of cyclin-dependent kinase inhibitor, p21Waf1/Cip1. The underlying mechanism of the noted biological processes include KDM3A-mediated stimulation of Sox2 expression, and demethylating p53 protein and consequently, modulating its target genes such as Bcl-2 and p21Waf1/Cip1 expression. Consistently, KDM3A depletion inhibited the growth of subcutaneously implanted cisplatin-resistant human ovarian cancer cells in athymic nude mice. Moreover, KDM3A is abundantly expressed and positively correlated with Sox2 expression in human ovarian cancer tissues. In brief, our findings reveal a novel mechanism by which KDM3A promotes ovarian CSCs, proliferation and chemoresistance and thus, highlights the significance of KDM3A as a novel therapeutic target for resistant ovarian cancer.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Phenol-Explorer: an online comprehensive database on polyphenol contents in foods.

A number of databases on the plant metabolome describe the chemistry and biosynthesis of plant chemicals. However, no such database is specifically focused on foods and more precisely on polyphenols, one of the major classes of phytochemicals. As antioxidants, polyphenols influence human health and may play a role in the prevention of a number of chronic diseases such as cardiovascular diseases, some cancers or type 2 diabetes. To determine polyphenol intake in populations and study their association with health, it is essential to have detailed information on their content in foods. However this information is not easily collected due to the variety of their chemical structures and the variability of their content in a given food. Phenol-Explorer is the first comprehensive web-based database on polyphenol content in foods. It contains more than 37,000 original data points collected from 638 scientific articles published in peer-reviewed journals. The quality of these data has been evaluated before they were aggregated to produce final representative mean content values for 502 polyphenols in 452 foods. The web interface allows making various queries on the aggregated data to identify foods containing a given polyphenol or polyphenols present in a given food. For each mean content value, it is possible to trace all original content values and their literature sources. Phenol-Explorer is a major step forward in the development of databases on food constituents and the food metabolome. It should help researchers to better understand the role of phytochemicals in the technical and nutritional quality of food, and food manufacturers to develop tailor-made healthy foods. Database URL: http://www.phenol-explorer.eu.

Predicting subcellular localization of proteins using machine-learned classifiers.

MOTIVATION: Identifying the destination or localization of proteins is key to understanding their function and facilitating their purification. A number of existing computational prediction methods are based on sequence analysis. However, these methods are limited in scope, accuracy and most particularly breadth of coverage. Rather than using sequence information alone, we have explored the use of database text annotations from homologs and machine learning to substantially improve the prediction of subcellular location. RESULTS: We have constructed five machine-learning classifiers for predicting subcellular localization of proteins from animals, plants, fungi, Gram-negative bacteria and Gram-positive bacteria, which are 81% accurate for fungi and 92-94% accurate for the other four categories. These are the most accurate subcellular predictors across the widest set of organisms ever published. Our predictors are part of the Proteome Analyst web-service.

CA125 levels are a weak predictor of optimal cytoreductive surgery in patients with advanced epithelial ovarian cancer.

The utility of preoperative CA125 to predict optimal primary tumor cytoreduction in patients with advanced (stages IIIC and IV) epithelial ovarian cancer is controversial. In this paper, we retrospectively review patients with stage IIIC and IV epithelial ovarian cancer who underwent primary cytoreductive surgery from 1989 to 2001. Ninety-nine patients were identified and included in the analysis. All patients had preoperative CA125 levels measured. Operative and pathology reports were reviewed. Optimal cytoreduction was defined as largest volume of residual disease < 1 cm in maximal dimension. Mean values were compared with t-test on a log scale when needed. The optimal cut-point for discriminating between those with vs. without optimal cytoreduction was determined using the receiver operator curve (ROC) method. Optimal cytoreduction was achieved in 73% of patients. Among patients with optimal cytoreductive status the mean CA125 level was 569, while among patients with suboptimal cytoreduction the mean CA125 level was 1520 (P < 0.007). A CA125 level of 912 was identified as the optimal cut-point to distinguish the two groups. Using this CA125 level, the sensitivity of this test in predicting optimal cytoreduction was 58% and the specificity was 54%. The positive predictive value of CA125 for optimal cytoreduction was 78% and the negative predictive value was 31%. We conclude that CA125 level is a weak positive and negative predictor of optimal cytoreductive surgery in patients with advanced epithelial ovarian cancer. The CA125 level should not be used as a primary predictor of the outcome of cytoreductive surgery and should be viewed in the context of all other preoperative features.

FIGO stage III and IV uterine papillary serous carcinoma: impact of residual disease on survival.

The objective of this study was to assess the impact of surgical cytoreduction on the survival of patients with uterine papillary serous carcinoma (UPSC). Patients added to the institutional tumor registries between January 1980 and September 2001 with the diagnosis of UPSC were reviewed. The records of 43 patients who underwent surgical cytoreduction for FIGO stage III and IV disease were reviewed. The median survival of UPSC patients with microscopic residual disease was significantly improved compared to those with macroscopic residual disease following primary surgical cytoreduction. We conclude that primary surgical cytoreduction resulting in microscopic residual disease is associated with an improvement in recurrence-free survival and overall survival in women with UPSC.

Differences due to collimator blurring in cardiac images with use of circular and elliptic camera orbits.

BACKGROUND: In cardiac imaging systems, an elliptic acquisition orbit about the patient can be used to enhance resolution of single photon emission computed tomography (SPECT) images by minimizing the distance between the object imaged and the rotating detector system. In this study artifacts from images acquired with the standard circular acquisition are compared with those acquired with various elliptic acquisitions. METHODS AND RESULTS: With the use of elliptic camera orbits of different eccentricities, simulated projection data were generated from a slice through the left ventricle (LV). The projection data included a simulation of the degradation due to the depth-dependent response of the collimator. As is common in many clinical systems, SPECT images were reconstructed with the standard filtered backprojection algorithm without correction for the collimator response. When the ratio of the major-to-minor axis of the acquisition arc is changed from 1 (circular) to 1.5 (elliptic), reconstructed SPECT images show an additional loss of counts (about 10%) in the apical region of the LV. The severity of the apical defect is also dependent on the starting angle of the acquisition arc. When the starting angle is changed from 0 degrees (detector parallel to the major axis of the LV) to 60 degrees, the ratio between the minimum count in the apical region and the maximum count in the left ventricular myocardial wall decreases by as much as 20%. CONCLUSIONS: SPECT image artifacts from elliptic acquisitions are significantly more severe than those from circular acquisitions. Because of the significant difference in images reconstructed from circular and elliptic acquisitions, standardized normal files acquired from circular acquisitions should not be used for comparisons with patient data acquired from elliptic acquisitions.

NID67, a small putative membrane protein, is preferentially induced by NGF in PC12 pheochromocytoma cells.

In an effort to identify genes involved in neuronal differentiation, we have used representational difference analysis (RDA) to clone cDNAs that are preferentially induced by nerve growth factor (NGF) vs. epidermal growth factor (EGF) in PC12 pheochromocytoma cells. We now report the cloning of a previously unknown primary response gene, NID67. In addition to a robust induction by NGF and FGF, both of which cause PC12 cells to differentiate, NID67 is strongly induced by forskolin, A23187 and ATP. EGF, TPA and KCl induce NID67 only weakly. NID67 mRNA is most abundant in heart, ovary and adrenal. Modest levels are present in most brain regions, testis, thyroid, thymus, pituitary, kidney and intestine; little NID67 is present in skeletal muscle and cerebellum. The NID67 cDNA contains a 180 bp open reading frame (ORF) that encodes a 60 amino acid protein. The central 29 amino acids are very hydrophobic and very likely comprise a transmembrane domain. Mouse and human NID67 cDNAs contain an ORF similar to NID67; the rat and human protein sequences are 85% identical whereas the rat and mouse sequences are 92% identical. In vitro transcription and translation reactions confirmed that the ORF we identified produces a 6000 Da protein product. Several small membrane proteins are similar to NID67; they contain a transmembrane domain and little more. All of these proteins participate in forming or regulating ion channels. NID67 may play a similar role in cellular physiology.

Expression of the urokinase plasminogen activator receptor is transiently required during "priming" of PC12 cells in nerve growth factor-directed cellular differentiation.

We previously identified the urokinase plasminogen activator receptor (UPAR) as a gene induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF), in PC12 cells (Farias-Eisner et al. [2000] J. Neurosci. 20:230-239). Antisense oligonucleotides for the UPAR mRNA or an antibody directed against UPAR protein, added simultaneously with NGF, block NGF-induced morphological and biochemical differentiation of PC12 cells. In this report, we show that anti-UPAR antibody blocks morphological differentiation and the expression of two NGF-specific secondary response genes, collagenase-1 and transin, in PC12 cells only during the first 2 hr following NGF exposure. These data suggest that induced UPAR expression is required only over a short period of time following exposure to NGF for the differentiation program in PC12 cells to proceed. For two models of "primed" PC12 cells, we found that UPAR expression and function are not required for NGF-induced differentiation. UPAR and the secondary response genes collagenase-1 and transin are not induced in "primed" PC12 cells in response to NGF, and anti-UPAR antibody does not block morphological differentiation in these cells. Our data suggests that UPAR is required only transiently during the "priming" of PC12 cells in NGF-induced PC12 cell differentiation.

Searching for depolarization-induced genes that modulate synaptic plasticity and neurotrophin-induced genes that mediate neuronal differentiation.

We identify and characterize two classes of immediate-early genes: (i) genes, induced by depolarization in neurons, that play a role in depolarization-induced neuronal plasticity and (ii) genes, induced in neuronal precursors by neurotrophins, that play a causal role in neurotrophin-directed neuronal differentiation. We use rat PC12 pheochromocytoma cells to identify (i) genes preferentially induced by [depolarization or forskolin] versus [Nerve Growth Factor (NGF) or Epidermal Growth Factor (EGF)] and (ii) genes preferentially induced by NGF versus EGF. We describe (i) a collection of genes preferentially induced by depolarization/forskolin in PC12 cells and by kainic acid in vivo, and (ii) a collection of genes preferentially induced by NGF. The synaptotagmin IV gene encodes a synaptic vesicle protein whose level is modulated by depolarization. NGF preferentially induces the urokinase-plasminogen activator receptor in PC12 cells. Antisense oligonucleotide and anti-UPAR antibody experiments demonstrate that NGF-induced UPAR expression is required for NGF-driven PC12 cell differentiation.

The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

Cancer of the uterine cervix.

The overall five-year survival of cervical cancer is only 40% worldwide despite the development of effective screening modalities. Paramount to this issue is access to appropriate medical care which remains limited to high-risk populations throughout the world. While surgery and radiation remain the mainstays of current treatment, new therapies based on the association of cervical cancer with the human papillomavirus are currently under investigation.

Dysgerminoma: the role of conservative surgery.

Twenty-five cases of pure ovarian dysgerminoma treated at UCLA Medical Center between 1958 and 1992 were reviewed retrospectively. Patterns of recurrence and overall survival were analyzed with regard to primary surgery (conservative versus nonconservative), use of adjuvant therapy, and stage of disease. Fourteen patients (56%) underwent conservative surgical therapy defined as preservation of the contralateral ovary, 10 patients (40%) had nonconservative primary surgery, and one patient (4%) had chemotherapy as primary treatment. Three patients (12%) received adjuvant chemotherapy and nine patients (36%) received postoperative radiation therapy. Fifteen patients (60%) had stage I disease, four (16%) stage II, and three each (12%) had stage III and IV disease. Nine patients (36%) experienced recurrence of disease. Seven of these nine patients (78%) had stage I disease and all seven had undergone conservative primary surgery with preservation of the contralateral ovary. Six of the seven had received no adjuvant therapy. Only one of these seven patients experienced recurrence in the preserved ovary. She was found to have a dysgenetic ovary and an XY karyotype. Three patients with recurrent disease had received radiation therapy after primary surgery. Twenty patients (80%) were alive without disease at follow-up, two patients (8%) were alive with disease, and three (12%) had died of disease. There was no statistically significant difference in recurrence rates between those patients treated with conservative surgery and those treated with nonconservative surgery, although the total number of patients with recurrences was greater in the former group. Our data suggest that a conservative surgical approach is the preferred treatment in patients with pure dysgerminoma of the ovary who desire future fertility. Lack of adjuvant chemotherapy or radiation therapy, rather than type of initial surgery, may be associated with a higher risk of recurrence.

Attenuation artifacts in SPECT: effect of wrap-around lung in 180 degrees cardiac studies.

UNLABELLED: We estimated that in 75%-90% of PET 82Rb patients the left lung appeared to wrap around the anterior aspect of the left ventricle. We used clinical PET 82Rb myocardial perfusion studies as the input to a SPECT computer simulation model to determine if patients with left lung wrap-around displayed consistent artifacts in reconstructed SPECT images. In particular, we sought an explanation for the hot lateral wall seen in SPECT images from normal female and male patients. METHODS: Attenuated SPECT 201Tl emission data were simulated from a mid-ventricular slice in 10 randomly selected clinical PET 82Rb studies with left lung wrap-around. In these same cases, the influence of left lung wrap-around was removed by assigning the left lung an attenuation coefficient which matched that of the heart. Five randomly selected clinical PET 82Rb studies without left lung wrap-around were also processed with our model. RESULTS: In all 10 cases with left lung wrap-around, reconstructed SPECT images showed the hot lateral wall artifact with a mean septal-to-lateral wall count ratio of 0.86. With left lung wrap-around removed in the same 10 patients, reconstructed images did not show hot lateral wall (mean septal-to-lateral wall count ratio = 1.07). The 5 cases without left lung wrap-around did not show hot lateral wall (mean septal-to-lateral wall count ratio = 1.04) and the ratios changed little with the filling of the left lung (mean septal-to-lateral wall count ratio = 1.05). CONCLUSION: Results of our PET-to-SPECT computer simulation model showed that the hot lateral wall artifact found in SPECT myocardial perfusion images was related to the orientation and positions of the left ventricle and the left lung.

The chemistry and tumoricidal activity of nitric oxide/hydrogen peroxide and the implications to cell resistance/susceptibility.

The mechanism of cytotoxicity of the NO donor 3-morpholino-sydnonimine toward a human ovarian cancer cell line (OVCAR) was examined. It was found that the NO-mediated loss of cell viability was dependent on both NO and hydrogen peroxide (H2O2). Somewhat surprisingly, superoxide (O2) and its reaction product with NO, peroxynitrite (-OONO), did not appear to be di- rectly involved in the observed NO-mediated cytotoxicity against this cancer cell line. The toxicity of NO/H2O2 may be due to the production of a potent oxidant formed via a trace metal-, H202-, and NO-dependent process. Because the combination of NO and H2O2 was found to be particularly cytotoxic, the effect of NO on cellular defense mechanisms involving H2O2 degradation was investigated. It was found that NO was able to inhibit catalase activity but had no effect on the activity of the glutathione peroxidase (GSHPx)-glutathione reductase system. It might therefore be expected that cells that utilize primarily the GSHPx-glutathione reductase system for degrading H2O2 would be somewhat resistant to the cytotoxic effects of NO. Consistent with this idea, it was found that ebselen, a compound with GSHPx-like activity, was able to protect cells against NO toxicity. Also, lowering endogenous GSHPx activity via selenium depletion resulted in an increased susceptibility of the target cells to NO-mediated toxicity. Thus, a possible NO/H2O2/metal-mediated mechanism for cellular toxicity is presented as well as a possible explanation for cell resistance/susceptibility to this NO-initiated process.

What is the role of reassessment laparoscopy in the management of gynecologic cancers in 1995?

One hundred fifty-four patients with a diagnosis of ovarian, primary peritoneal, or fallopian tube carcinoma underwent 181 reassessment procedures to detect persistent or recurrent disease between January 1, 1989 and December 31, 1994 at Cedars-Sinai Medical Center. One hundred four laparoscopic procedures were performed. Eleven of these procedures were converted to laparotomy due to severe adhesions. Therefore, a total of 88 reassessment laparotomies were performed during the study period. Fifty-seven of 93 laparoscopies and 69 of 88 laparotomies were done as second-look procedures. There was no significant difference between the two groups with respect to patient age, tumor histology, degree of primary cytoreduction, and tumor stage or grade. Significant differences were found between laparoscopy and laparotomy groups in the following outcome variables evaluated: estimated blood loss (33.9 ml vs 164.9 ml, P = 0.0001), operative time (81.3 min vs 130.4 min, P = 0.0001), days of hospitalization (0.3 days vs 6.8 days, P = 0.0001), and direct cost/case ($2765 vs $5420, P = 0.0001). Despite obtaining 50% fewer biopsies with laparoscopy than laparotomy, the ability to detect disease was similar between these two groups: 47.3% vs 55.7% for all procedures and 52.6% vs 53.6% in the patients undergoing second-look procedures. Major complications in the laparoscopy group included transverse colon perforation (1), small bowel perforation (2), enterocutaneous fistula (1), and a retroperitoneal hematoma (1). Major complications in the laparotomy group included cystotomy (1), left ureteral injury (1), enterotomy (2), and SBO (4). Laparoscopy, when technically feasible, appears equally as effective as laparotomy in detecting persistent or recurrent malignant disease with less blood loss, less days spent in the hospital, less financial burden, and no increase in patient morbidity.

Diagnosis of coronary artery disease in women: roles of three dimensional imaging with magnetic resonance or positron emission tomography.

Diagnosis and assessment of coronary artery disease (CAD) is especially difficult in women. The history of chest discomfort and various noninvasive tests each have particular problems, which indicate the need to consider more accurate tests such as cardiac magnetic resonance imaging (MRI) and positron emission tomography (PET). MRI of cardiac function at rest and during dobutamine stress has good accuracy, and MR Myocardial perfusion imaging (MPI) with gadolinium DTPA looks promising. The most exciting MR method is cineangiography (MRA), which images blood flow through the coronary arterial lumen as an intense signal. In an initial clinical trial this method showed excellent sensitivity and fair specificity in patients in whom adequate images could be obtained. MR spectroscopy (MRS) has imaged changes in high energy phosphates in patients with severe coronary stenoses during handgrip exercise, but is still experimental. PET MPI corrects the images for attenuation problems that limit the use of other radionuclide imaging procedures in women more than in men. Many studies show excellent sensitivity and specificity to diagnose CAD by PET MPI. In view of its clinical validation and the safety of dipyridamole relative to dobutamine, PET MPI appears to be the best test for assessing CAD in women. The greater accuracy of PET (or perhaps of fully developed MRI/MRA systems) will produce better clinical outcomes and cost-effectiveness for most patients than will less accurate modalities, despite their higher initial cost.

Endocervical sampling by Kevorkian curette or Pipelle aspiration device: a randomized comparison.

OBJECTIVE: Our purpose was to compare two means of endocervical sampling-the Kevorkian curette and the Pipelle aspiration device (Unimar Co., Wilton, Conn.)-with respect to patient discomfort, tissue volume, and specimen adequacy for diagnosis. STUDY DESIGN: Fifty-two women undergoing investigation of abnormal cervical cytologic results were assigned randomly to endocervical sampling by Kevorkian or Pipelle instruments. Pain associated with the procedure was assessed by having each subject indicate her pain level on a visual analog scale. Tissue volume was graded by examination of the microscopic slides by two investigators blinded to assignment. Adequacy for diagnosis was evaluated by reviewing pathology reports. RESULTS: Subjects having Pipelle endocervical aspiration (n = 24) had significantly lower mean (+/- SEM) pain scores (27 +/- 5 vs 48.5 +/- 7, p = 0.02) than those in whom the Kevorkian instrument was used (n = 28). However, there were no significant differences in tissue volume obtained or in proportions considered adequate for diagnosis. CONCLUSIONS: Use of the Pipelle instrument was associated with less patient discomfort than Kevorkian curettage for endocervical sampling while providing similar tissue volume and adequacy for diagnosis.