Keng Shen

R-loop-mediated genomic instability is caused by impairment of replication fork progression

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

Characterization and proteomic analysis of ovarian cancer-derived exosomes

Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, because most patients present with advanced disease at diagnosis. Exosomes are important intercellular communication vehicles, released by various cell types. Here we presented firstly the protein profile of highly purified exosomes derived from two ovarian cancer cell lines, OVCAR-3 and IGROV1. The exosomes derived from ovarian cancer cell lines were round and mostly 30-100 nm in diameter when viewed under an electron microscope. The exosomal marker proteins TSG101 and Alix were detected in exosome preparations. The range of density was between 1.09 g/ml and 1.15 g/ml. A total of 2230 proteins were identified from two ovarian cell-derived exosomes. Among them, 1017 proteins were identified in both exosomes including all of the major exosomal protein markers. There were 380 proteins that are not reported in the ExoCarta database. In addition to common proteins from exosomes of various origins, our results showed that ovarian cancer-derived exosomes also carried tissue specific proteins associated with tumorigenesis and metastasis, especially in ovarian carcinoma. Based on the known roles of exosomes in cellular communication, these data indicate that exosomes released by ovarian cancer cells may play important roles in ovarian cancer progression and provide a potential source of blood-based protein biomarkers.

Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome

Cancer stem cells (CSCs) play an important role in the recurrence and drug resistance of cancer. Isolation and characterization of CSCs from ovarian cancer samples may help to provide novel diagnostic and therapeutic targets in the management of recurrent disease and drug resistance in ovarian cancer. Here, we developed a xenograft model in which cells from 14 samples of human ovarian serous adenocarcinoma tissue or ascites were implanted in immunodeficient mice to test the tumorigenic potential of different populations of ovarian cancer cells. We identified and isolated the tumorigenic cells as CD117(+)Lineage(-) from three different xenografts. As few as 10(3) cells with the CD117(+)Lineage(-) phenotype, which comprise <2% of the xenograft tumor cells, were able to regenerate tumors in a mouse model, a 100-fold increase in tumorigenic potential compared to CD117(-)Lineage(-) cells. The tumors that arose from purified CD117(+)Lineage(-) cells reproduced the original tumor heterogeneity and could be serially generated, demonstrating the ability to self-renew and to differentiate, two defining properties of stem cells. Furthermore, immunohistochemistry analysis of 25 patients with advanced ovarian serous adenocarcinoma revealed positive immunostaining for CD117 in 40% (10 of 25) of patients. CD117 expression was statistically correlated with resistance to conventional chemotherapy (P=0.027). In conclusion, our study demonstrates that human ovarian cancer cells with the CD117(+) phenotype possess the unique properties of CSCs, including self-renewal, differentiation, a high tumorigenic potential, and chemoresistance. Future studies designed to target CD117(+) cancer cells may identify more attractive and effective therapies for treatment of ovarian cancer.

Preoperative neutrophil-lymphocyte and platelet-lymphocyte ratios as independent predictors of cervical stromal involvement in surgically treated endometrioid adenocarcinoma

Background The purpose of this study was to evaluate the relationship between preoperative inflammatory markers (neutrophil-lymphocyte ratio and platelet-lymphocyte ratio) and cervical stromal involvement in patients with endometrioid adenocarcinoma. Methods We studied 318 patients with endometrioid adenocarcinoma who underwent comprehensive surgical staging. We used univariate and multivariate analyses of cervical stromal involvement and receiver-operating curves to calculate optimal cutoff values for neutrophil-lymphocyte and platelet-lymphocyte ratios to predict cervical stromal involvement. Results The presence of cervical stromal involvement was associated with neutrophil-lymphocyte ratio and platelet-lymphocyte ratio (P = 0.009 and P = 0.031, respectively). Multivariate analysis showed that higher neutrophil-lymphocyte and platelet-lymphocyte ratios independently predicted cervical stromal involvement (odds ratio 3.10, 95% confidence interval 1.10–8.76, P = 0.032, and odds ratio 5.27, 95% confidence interval 1.94–14.35, P = 0.001, respectively). At a threshold of 2.01, the neutrophil-lymphocyte ratio was 71.0% sensitive and 63.8% specific for stromal involvement; at a 172.24 threshold, the platelet-lymphocyte ratio was 48.4% sensitive and 88.9% specific. Conclusion Preoperative neutrophil-lymphocyte and platelet-lymphocyte ratios can help identify the risk of cervical stromal involvement in patients with endometrial cancer. Evaluating these ratios may help select patients who should be particularly watched and tested for cervical stromal involvement.

A clinicopathological analysis of 40 cases of ovarian Sertoli-Leydig cell tumors

OBJECTIVE To evaluate the clinicopathological features of ovarian Sertoli-Leydig cell tumors (SLCTs) and to explore the reasonable therapy. METHODS A total of 40 cases of SLCTs were retrospectively reviewed. RESULTS The incidence of SLCTs was 0.41%, with a median age of 28 years. All tumors were confined to one ovary. Four tumors were well differentiated, 14 were intermediately differentiated, 20 were poorly differentiated, and 2 were undefined; 2 cases had heterologous elements, and 3 had a retiform pattern. The patients were classified into 3 groups: androgen excess (25/40), estrogen excess (6/40), and no endocrine changes (9/40). The percentages of tumors >10 cm in diameter were 8.0%, 16.7% and 40.0%, respectively; the percentages of poor differentiation were 40.0%, 50.0% and 77.8%, respectively; and the percentages of tumor rupture were 20.0%, 16.7% and 66.7%, respectively. One patient underwent cystectomy, 27 underwent unilateral salpingo-oophorectomy, and 12 underwent total hysterectomy and bilateral salpingo-oophorectomy. A total of 23 patients received postoperative chemotherapy. One patient died of diabetic nephropathy, and 3 were lost to follow up. The remaining 36 were followed up from 12 to 377 (average 70.4) months. Two patients with stage Ic tumors of poor differentiation had a recurrence within 13 and 21 months, and both obtained complete remission after the second surgery and chemotherapy. CONCLUSIONS The prognosis of SLCTs is good, although poorly differentiated tumors may recur. Conservative surgery is acceptable for young patients wishing to preserve fertility, and postoperative adjuvant chemotherapy and long-term follow up are recommended to those with high-risk factors.

Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials

Objective To identify differentially expressed long non-coding RNAs (lncRNAs) involved in the metastasis of epithelial ovarian cancer. Methods An in vitro invasion assay was performed to validate the invasive capability of SKOV3 and SKOV3.ip1 cell lines. Total RNA was then extracted, and microarray analysis was performed. Moreover, nine lncRNAs were selected for validation using RT-qPCR. Results Compared with the SKOV3 cells, the SKOV3.ip1 cells significantly improved in the in vitro invasive activity. Of the 4,956 lncRNAs detected in the microarray, 583 and 578 lncRNAs were upregulated and downregulated, respectively, in SKOV3.ip1 cells, compared with the parental SKOV3 cells. Seven of the analyzed lncRNAs (MALAT1, H19, UCA1, CCAT1, LOC645249, LOC100128881, and LOC100292680) confirmed the deregulation found by microarray analysis. Conclusion LncRNAs clusters were differentially expressed in ovarian cancer cells with varying metastatic potentials. This result indicates that some lncRNAs might exert a partial or key role in epithelial ovarian cancer metastasis. Further studies should be conducted to determine the roles of these lncRNAs in ovarian cancer metastasis.

A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA

OBJECTIVE Epithelial ovarian cancer (EOC) remains the most lethal disease among gynecological malignancies. Prompt diagnosis is challenging because of the non-specific symptoms exhibited during the early stage of the disease. As a result, there is an urgent need for improved detection methods. In this study, we established a multiplex methylation-specific PCR (MSP) assay to improve the early detection of ovarian cancer, via identification of the methylation status of cell-free serum DNA. METHODS After screening, we chose seven candidate genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5 and OPCML) with a high frequency of methylation to construct the multiplex-MSP assay. When methylation of at least one of the seven genes was observed, the multiplex-MSP assay was considered positive. We performed retrospective and screening studies to verify the specificity and sensitivity of the assay in the detection of EOC. RESULTS The methylation status of cell-free serum DNA was examined in the preoperative serum of 202 patients, including 87 EOC patients (stage I, n=41; stage II-IV, n=46), 53 patients with benign ovarian tumors and 62 healthy controls. As expected, the multiplex MSP assay achieved a sensitivity of 85.3% and a specificity of 90.5% in stageI EOC, strikingly higher rates compared with a single CA125, which produced a sensitivity of 56.1% at 64.15% specificity [P=0.0036]. CONCLUSION A multiplex MSP assay that analyzes the methylation status of cell-free serum DNA is a suitable and reliable approach to improve the early detection of ovarian cancer, potentially benefiting a broad range of applications in clinical oncology.

Clinical analysis of ovarian epithelial carcinoma with coexisting pelvic endometriosis

OBJECTIVES To explore the differences between women with endometriosis associated ovarian cancer and typical epithelial ovarian cancer. STUDY DESIGN The medical charts of total 226 patients with epithelial ovarian cancer treated at Peking Union Medical College Hospital between March 2011 and March 2012 were reviewed. Histology evaluation determined endometriosis associated ovarian cancer (n = 17) or non-endometriosis associated ovarian cancer (n = 209). RESULTS Compared with non-endometriosis associated ovarian cancer, patients with endometriosis associated ovarian cancer were proved: (1) to be younger and more likely to be premenopausal at diagnosis of epithelial ovarian cancer (P = .03 and .005, respectively); (2) to have lower preoperative serum level of Ca125 (mean: 122.9 vs 1377.5 U/mL, P < .001) and more likely to display normal Ca125 level (P < .001); (3) to be identified at the earlier stage (stage I, P < .001); (4) to have completely different distribution of histological subtypes (significant overrepresentation of clear cell and endometrioid carcinoma). CONCLUSION As such, patients with endometriosis associated ovarian cancer differ from non-endomertiosis associated ovarian cancer in many of their critical clinical and biologic characteristics.

The PI3K/AKT/mTOR pathway is a potential predictor of distinct invasive and migratory capacities in human ovarian cancer cell lines

Objectives To explore the genetic and molecular events that control subclones exhibiting distinct invasive/migratory capacities derived from human epithelial ovarian cancer (EOC) cell line A2780 and SKOV3. Methods Single-cell subclones were isolated and established that were derived from the SKOV3 and A2780 cell lines through limiting dilution methodology. Transwell insert assays and MTT assays were performed to screen and identify the subclones exhibiting the highest and the lowest invasive/migratory capacities, and the selected subclones were renamed as A-H (A2780 high), A-L (A2780 low), S-H (SKOV3 high), and S-L (SKOV3 low). Their biological characteristics were evaluated. RNA-Seq was conducted on the targeted subclones. Results Compared with their corresponding counterparts, A-H/S-H cells exhibited significantly higher invasive/migratory capacities (P < 0.001 and = 0.001, respectively). A-H/S-H cells displayed a clear reduction in doubling time (P = 0.004 and 0.001, respectively), and a significant increase in the percentage of cells in S phase (P = 0.004 and 0.022, respectively). Additionally, the apoptotic rates of A-H/S-H cells were significantly lower than those of A-L/S-L cells (P = 0.002 and 0.026, respectively). At both mRNA and protein levels, caspase-3 and caspase-7 expression were reduced but Bcl-2 expression was increased in A-H/S-H cells. The TrkB (anoikis-related) and Beclin1 (autophagy-related) levels were consistently high and low, respectively, in both A-H/S-H cells. Resistance to chemotherapy in vitro and higher capacities on tumor formation in vivo was presented in both A-H/S-H cells. PI3K/AKT/mTOR pathway components, PIK3CA, PIK3CD, AKT3, ECM1, GPCR, mTOR and PRKCB were increased but that the Nur77 and PTEN were decreased in A-H/S-H cells, identified by RNA-Seq and consistently confirmed by RT-PCR and Western blot analyses. Conclusions Heterogeneous cell subpopulations exhibiting distinct invasive and migratory capacities co-exist within the SKOV3 and A2780 cell lines. PI3K/AKT/mTOR pathway activation is associated with higher invasive and migratory capacities in subpopulations of human ovarian cancer cell lines. Inhibiting this pathway may be useful for the chemoprevention or treatment of EOC.

A systematic review of quality of life and sexual function of patients with cervical cancer after treatment.

Objectives Quality of life (QoL) and sexual health have become increasingly important in cervical cancer survivors (CCSs). The aims of this review were to summarize research findings of QoL and sexual function in CCSs after treatment on the basis of self-reported questionnaires and to update the current knowledge of overall QoL and sexual function in CCSs. Methods Studies from electronic database between May 1966 and May 2013 were rated on their internal validity as methodological assessment. Thirty-two studies were included, wherein 15 studies had a relatively good methodology. Results Anorectal function, urinary symptoms, and lymphedema were commonly reported as physical symptoms. As to psychosocial domains, the studies illustrated that anxiety decreased with age, whereas depression generally increased with age. Sexual function was involved in most of the studies. Vaginal dryness, dyspareunia, short vagina, and sexual dissatisfaction were prominent issues of sexual dysfunction and vaginal changes in CCSs. In terms of treatment modality, radiotherapy was thought to be associated with worse QoL and sexual function in CCSs. Conclusions The studies showed that QoL and sexual function in CCSs were compromised compared with the general population to different extents. Quality of life and sexual function should be paid with more attention in patients with cervical cancer after treatment.