Identification of negative regulators of p53 pathway by a forward genetic screen in ovarian cancer

Abstract Ovarian cancer is the deadliest of all female gynecological cancers and the fifth leading cause of cancer-related death in women. Lack of early detection and treatment failure are the major contributors to ovarian cancer death in women. Understanding ovarian cancer at the molecular level will help provide better treatment options, such as targeted therapies. Nearly sixty percent of women are diagnosed at late stage ovarian cancer. TP53 is the most commonly mutated gene in ovarian cancer as well as in all other cancers. However, there are subsets of ovarian cancer patients with wild-type TP53, and we would like to understand the molecular and cellular mechanisms that disable the transcriptional functions of p53 in those patients. Wild-type p53 is the transcription factor that activates sets of genes to respond to various types of cellular and molecular stress and to maintain genomic stability in normal cells. The major function of wild-type p53 is the transcriptional activation of genes that are involved in cell cycle arrest to repair DNA damage and cell death if the damage is beyond repair in cells. However, the p53 transcriptional functions of cell cycle arrest and cell death are not observed in those ovarian cancer patients with wild-type TP53 and the genes inhibiting transcriptional activities of p53 are not well studied. Additionally, our understanding of negative regulators of p53 in cell activity is not complete, and identifying those regulators may provide new insights into how the p53 pathway can be deregulated in tumors without mutations in TP53. For that purpose, we performed forward genetic screening using a patient-derived pool cDNA library constructed with pRetro-LIB vector in wild-type TP53 ovarian cancer cells to identify the potential negative regulators of p53. First, we tested our method of screening in wild-type TP53 ovarian cancer cells to prove the concept of our experimental approach. We found that using modified 293T cells (Phoenix AMPHO) with spin-fection method overcame the low transfection efficiency of retroviral particles in wild-type TP53 ovarian cancer cells (A2780). In normal cells, p53 is present at relatively low levels because of the negative feedback loop between Mdm2 and p53. To induce the p53 level in ovarian cancer cells with wild-type TP53, we used the small molecule inhibitor, Nutlin-3a. Nutlin-3a blocks the interaction between Mdm2 and p53, thereby allowing the stabilization of p53 in ovarian cancer cells. Stabilized p53 transactivates genes that initiate cell cycle arrest or cell death. Accordingly, Nutlin-3a suppresses the growth of cancer cells with wild-type TP53. We used this system to screen for exogenous genes from the patient-derived cDNA library that block Nutlin-3a-mediated growth suppression in A2780 cancer cells. The screen identified three candidate genes (NIFK, GXYLT 1, and SACS) that prevent the Nutlin-3a-induced growth suppression in A2780 cancer cells, and NIFK (also known as Nucleolar Protein Interacting with the FHA Domain of MKI67 or MKI67IP) was identified in four independent screening experiments. NIFK encodes a protein that interacts with the forkhead-associated domain of the Ki-67 antigen and has been implicated in ribosome biogenesis, e.g., pre-ribosomal RNA processing and ribosome assembly. NIFK also associates with pre-mRNAs. However, the potential association between NIFK and p53 in ovarian cancer is unknown. We evaluated the level of NIFK, p53, and Mdm2 in NIFK-overexpressing ovarian cancer cells. We found that the dose-dependent effect of NIFK expression in preventing the Nutlin-3a-induced growth suppression in A2780 cells. Our results suggest that NIFK overcomes the growth arrest by functional p53 induced by Nutlin-3a, and NIFK negatively regulates p53 pathway in ovarian cancer cells. In summary, our studies showed that NIFK overexpression cells play a role in cell survival, proliferation and the negative regulation of p53 in ovarian cancer cells, and this should be further explored to understand the molecular function of NIFK

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion

Xanthogranulomatous epithelial tumor: report of 6 cases of a novel, potentially deceptive lesion with a predilection for young women.

Epithelial marker expression and/or epithelial differentiation, as well as "anomalous" expression of keratins, are features of some soft tissue tumors. Recently, we have encountered an unusual mesenchymal tumor composed of bland, distinctly eosinophilic, keratin-positive epithelial cells, which were almost entirely obscured by xanthogranulomatous inflammation. Six cases were identified (5 F, 1 M; 16-62 years (median 21 years)) arising in soft tissue (n = 4) and bone (n = 2) and ranging in size from 2 to 7 cm. The tumors were generally circumscribed, with a fibrous capsule containing lymphoid aggregates, and consisted in large part of a sheet-like proliferation of foamy histiocytes, Touton-type and osteoclast-type giant cells, and chronic inflammatory cells. Closer inspection, however, disclosed a distinct population of uniform, cytologically bland mononuclear cells with brightly eosinophilic cytoplasm arranged singly and in small nests and cords. Overt squamous and/or glandular differentiation was absent. By immunohistochemistry, these cells were diffusely positive with the OSCAR and AE1/AE3 keratin antibodies, and focally positive for high-molecular weight keratins; endothelial and myoid markers were negative and SMARCB1 was retained. RNA-seq identified a PLEKHM1 variant of undetermined significance in one case, likely related to this patient's underlying osteopetrosis. Follow-up to date has been benign. In summary, we have identified a novel tumor of soft tissue and bone with a predilection for young females, provisionally termed "xanthogranulomatous epithelial tumor". These unusual lesions do not appear to arise from adnexa, or represent known keratin-positive soft tissue tumors, and the origin of their constituent epithelial cells is obscure. The natural history of this distinctive lesion appears indolent, although study of additional cases and longer term follow-up are needed

Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis

High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification

GLR Test for OFDM System Identification Using Pilot Tones Pattern

International audienceIn the context of cognitive network architecture, an opportunistic cognitive receiver must identify the present active networks. In this paper, we propose an efficient algorithm for the identification of OFDM networks exploiting the pilot patterns used in these standards which are prescribed uniquely by their underlying standards. These pilots are inserted for the channel estimation and synchronization between the base stations and their users. The proposed Generalized Likelihood Ratio Test (GLRT) not only allows a cognitive observer to detect the active networks by analyzing the observed signals but also performs channel estimation, time-frequency synchronization as well as estimation of the noise variance. These informations are of a great interest for Quality of Service estimation in the purpose of an association with the base station. The proposed solution is applicable to the existing standards (e.g., LTE, WiMAX, WiFi), doesn’t require any signaling overhead to be embedded on the pilot tones, is computationally inexpensive and only requires to know the pilot patterns. An other GLRT is proposed as a pre-detector which ignores the pilot information and allows to reduce the computational cost of the system for the cases where a large number of patterns/systems are to be tested