NANO-SBT-PEDF delivery system: A promising approach against ovarian cancer?

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein involved in various biological processes. Its expression declines during ovarian carcinogenesis where it could decrease macrophages polarization, inhibit angiogenesis and induce apoptosis. Altogether, PEDF represents an ideal anti-cancer agent against ovarian cancer. We previously proposed the non-viral Sleeping Beauty transposon (SBT) system to stably integrate the PEDF transgene into ovarian cancer cells. Here, we report the development of liposomes and lipid nanoparticles for SBT-PEDF gene therapy. We determined that the SBT-PEDF nanolipid delivery system was the best system to increase the expression of PEDF in ovarian cancer spheroids. We also developed an ex vivo model of ovarian tumors which allowed us to show that nanolipoplexe in combination to paclitaxel exhibits synergistic and effective anti-tumor efficacy on ovarian tumors. These findings demonstrate that lipid nanoparticle for SBT-PEDF gene therapy may be a promising therapeutic approach for ovarian cancer.

A Cellular Assay for Spike/ACE2 Fusion: Quantification of Fusion-Inhibitory Antibodies after COVID-19 and Vaccination.

Not all antibodies against SARS-CoV-2 inhibit viral entry, and hence, infection. Neutralizing antibodies are more likely to reflect real immunity; however, certain tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies but are limited by biosafety and standardization issues. We have developed a Spike/ACE2-dependent fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Cell fusion occurred rapidly allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of proteases. Sera from COVID-19-negative, non-vaccinated individuals or from patients at the moment of first symptoms did not lead to a significant reduction of fusion. Sera from COVID-19-positive patients as well as from vaccinated individuals reduced the fusion. This assay was more correlated to pseudotyped-based entry assay rather than serology or competitive ELISA. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a complete Spike/ACE2 interaction active on entry with a high degree of standardization, easily allowing automation in a standard bio-safety environment.

Decreased Levels of SARS-CoV-2 Fusion-Inhibitory Antibodies in the Serum of Aged COVID-19 Patients.

Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the underlying mechanisms of the disease are still poorly understood. In this study, we investigated fusion inhibitory antibodies (fiAbs) in elderly and younger COVID-19 patients and analyzed predictive factors for their occurrence. Methods: Data and samples were collected in two cohorts of hospitalized patients. A fusion assay of SARS-CoV-2 spike-expressing cells with ACE2-expressing cells was used to quantify fiAbs in the serum of patients. Results: A total of 108 patients (52 elderly (mean age 85 ± 7 years); 56 young (mean age 52 ± 10 years)) were studied. The concentrations of fiAbs were lower in geriatric patients, as evidenced at high serum dilutions (1/512). The association between fiAbs and anti-Spike Ig levels was weak (correlation coefficient < 0.3), but statistically significant. Variables associated with fusion were the delay between the onset of symptoms and testing (HR = −2.69; p < 0.001), clinical frailty scale (HR = 4.71; p = 0.035), and WHO severity score (HR = −6.01, p = 0.048). Conclusions: Elderly patients had lower fiAbs levels after COVID-19 infection. The decreased fiAbs levels were associated with frailty.

Molecular Mechanisms of Trophoblast Dysfunction Mediated by Imbalance between STOX1 Isoforms.

STOX1 is a transcription factor involved in preeclampsia and Alzheimer disease. We show that the knock-down of the gene induces rather mild effect on gene expression in trophoblast cell lines (BeWo). We identified binding sites of STOX1 shared by the two major isoforms, STOX1A and STOX1B. Profiling gene expression of cells overexpressing either STOX1A or STOX1B, we identified genes downregulated by both isoforms, with a STOX1 binding site in their promoters. Among those, STOX1-induced Annexin A1 downregulation led to abolished membrane repair in BeWo cells. By contrast, overexpression of STOX1A or B has opposite effects on trophoblast fusion (acceleration and inhibition, respectively) accompanied by syncytin genes deregulation. Also, STOX1A overexpression led to abnormal regulation of oxidative and nitrosative stress. In sum, our work shows that STOX1 isoform imbalance is a cause of gene expression deregulation in the trophoblast, possibly leading to placental dysfunction and preeclampsia.

Malignant ascites: a source of therapeutic protein against ovarian cancer?

Ovarian cancer is the fifth leading cause of cancer-related death in the world. Some ovarian cancer patients present large amount of ascites at the time of diagnosis which may play an active role in tumor development. In earlier studies, we demonstrated that the acellular fraction of ascites can induce apoptosis of ovarian cancer cells. The current study identifies pigment epithelium derived factor (PEDF) as the molecule responsible for the apoptotic effect of ascites and evaluates the Sleeping Beauty transposon (SBT) system as a new tool for PEDF gene therapy against ovarian cancer. We utilize gel filtration, mass spectrometry, affinity column, cell viability assay, tumor development on chick chorioallantoic membrane and molecular biology techniques for these purposes. PEDF was thus identified as the agent responsible for the effects of ascites on ovarian cancer cell viability and tumor growth. Interestingly, the PEDF expression is decreased in ovarian cancer cells compared to healthy ovarian cells. However, the level of PEDF is higher in ascites than in serum of ovarian cancer patients suggesting that cells present in the tumor environment are able to secrete PEDF. We then used the SBT system to stably induce PEDF expression in ovarian cancer cells. The overexpression of PEDF significantly reduced the tumor growth derived from these cells. In conclusion, the results presented here establish that PEDF is a therapeutic target and that PEDF from ascites or SBT could be utilized as a therapeutic strategy for the treatment of ovarian cancer.

Single cell transcriptome in aneuploidies reveals mechanisms of gene dosage imbalance.

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.

The fine-tuning of endoplasmic reticulum stress response and autophagy activation during trophoblast syncytialization.

The syncytiotrophoblast (STB) is a multinuclear layer forming the outer surface of the fetal part of the placenta deriving from villous cytotrophoblastic cell (vCTB) fusion and differentiation. This syncytialization process is characterized by morphological and biochemical alterations of the trophoblast, which probably require removal of pre-existing structures and proteins to maintain cell homeostasis and survival. Interestingly, autophagy, which allows degradation and recycling of cellular components, was shown to be activated in syncytiotrophoblast. Here we examined the involvement of endoplasmic reticulum stress (ERS) response in autophagy activation during vCTB syncytialization. We first demonstrated the activation of ERS response and autophagy during the time course of trophoblastic cell fusion and differentiation. Alteration of autophagy activation in vCTB by chemical treatments or Beclin-1 expression modulation leads to a decrease in trophoblastic syncytialization. Furthermore, ERS response inhibition by chemical treatment or siRNA strategy leads to a default in syncytialization, associated with alteration of autophagy markers and cell survival. From these data, we suggest that ERS response, by fine regulation of autophagy activation, may serve as an adaptive mechanism to promote cell survival during trophoblastic syncytialization.

Extensive cellular heterogeneity of X inactivation revealed by single-cell allele-specific expression in human fibroblasts.

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.

Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours.

Biallelic mismatch repair deficiency (bMMRD) in tumours is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1, and results in a characteristic mutational profile. In this article, we describe the genetic basis of ultramutated high-grade brain tumours in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high-grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant, p.R802*, in the PMS2 gene. Additionally, by genome sequencing of these tumours, we found extremely high somatic mutation rates (237/Mb and 123/Mb), as well as somatic mutations in the proofreading domain of POLE polymerase (p.P436H and p.L424V), which replicates the leading DNA strand. Most interestingly, we found, in both cancers, that the vast majority of mutations were consistent with the signature of POLE exo- , i.e. an abundance of C>A and C>T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumour suppressor genes is more than two-fold lower in ultramutated tumours than in other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumours as being due to a combination of PMS2 germline and POLE somatic variants, and confirmed them as bMMRD/POLE exo- disorders. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comparative secretome of ovarian serous carcinoma: Gelsolin in the spotlight.

Ovarian cancer is one of the most common types of reproductive cancer, and has the highest mortality rate amongst gynecological cancer subtypes. The majority of ovarian cancers are diagnosed at an advanced stage, resulting in a five-year survival rate of ~30%. Early diagnosis of ovarian cancer has improved the five-year survival rate to ≥90%, thus the current imperative requirement is to identify biomarkers that would allow the early detection, diagnosis and monitoring of the progression of the disease, or of novel targets for therapy. In the present study, secreted proteins from purified ovarian control, benign and cancer cells were investigated by mass spectrometry, in order to identify novel specific markers that are easy to quantify in patients sera. A total of nine proteins revealed significant differential secretion from control and benign cells, in comparison with ovarian cancer cells. The mRNA expression levels of three of these proteins (Dickkopf protein 3, heat shock protein 10 kDa and gelsolin) were subsequently evaluated by reverse transcription-quantitative polymerase chain reaction. Combined with the protein level in serum, the present study identified that gelsolin may be a useful marker of ovarian cancer.

Detection of Imprinted Genes by Single-Cell Allele-Specific Gene Expression.

Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.

Circulating GRP78 antibodies from ovarian cancer patients: a promising tool for cancer cell targeting drug delivery system?

Glucose-regulated protein 78 (GRP78) is a chaperone protein that has a high frequency in tumor cells. Normally it is found in the endoplasmic reticulum to assist in protein folding, but under cellular stress, GRP78 influences proliferative signaling pathways at the cell surface. The increased expression elicits autoantibody production, providing a biomarker of ovarian cancer, as well as other types of cancer. This study aims to determine the epitope recognition of GRP78 autoantibodies isolated from serum of ovarian cancer patients and use the identified antibodies to design new drug delivery systems to specifically target cancer cells. We first confirmed that the membrane GRP78 levels are increased in ovarian cancer cells and positively correlate with proliferation. However, the level of circulating GRP78 autoantibodies did not correlate with membrane GRP78 expression in ovarian cancer cells and was lower, although not significantly, compared to control patients. We then determined the epitope recognition of GRP78 autoantibodies and showed that treatment with paclitaxel-loaded nanoparticles coated with anti-GRP78 antibodies significantly decreased tumor development in chick embryo culture of ovarian cancer cell tumors compared to paclitaxel treatment alone. This evidence suggests that nanoparticle drug delivery systems coupled with antibodies against GRP78 has potential as a powerful therapy against ovarian cancer.

Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10(-8)) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.

Role of PAR-4 in ovarian cancer.

Prostate apoptosis response-4 (PAR-4) is considered as a tumour suppressor due to its ability to selectively induce cell apoptosis in most cancer cells. However little is known about the role of PAR-4 in ovarian cancer. In this study, we investigated for the first time the role of PAR-4 in ovarian carcinogenesis. We showed that PAR-4 mRNA level is not significantly different between healthy and cancer ovarian cells. Immunohistochemistry on ovarian tissue showed that ovarian cancer cells are positive for PAR-4 nuclear and cytoplasmic staining whereas ovarian healthy cells are negative for PAR-4 nuclear staining. We then studied the role of PAR-4 in cell apoptosis. We determined that PAR-4 induces cell apoptosis in response to stimuli, in vitro, but is also involved in the relocation of GRP78 from endoplasmic reticulum to the cell surface of ovarian cancer cell line (SKOV-3 cells). In ovo, PAR-4 decreases ovarian tumour development and increases the response to taxol treatment. These observations suggest that PAR-4 is a very interesting therapeutic target against ovarian carcinogenesis.

Secretome Identifies Tenascin-X as a Potent Marker of Ovarian Cancer.

CA-125 has been a valuable marker for the follow-up of ovarian cancer patients but it is not sensitive enough to be used as diagnostic marker. We had already used secretomic methods to identify proteins differentially secreted by serous ovarian cancer cells compared to healthy ovarian cells. Here, we evaluated the secretion of these proteins by ovarian cancer cells during the follow-up of one patient. Proteins that correlated with CA-125 levels were screened using serum samples from ovarian cancer patients as well as benign and healthy controls. Tenascin-X secretion was shown to correlate with CA-125 value in the initial case study. The immunohistochemical detection of increased amount of tenascin-X in ovarian cancer tissues compared to healthy tissues confirms the potent interest in tenascin-X as marker. We then quantified the tenascin-X level in serum of patients and identified tenascin-X as potent marker for ovarian cancer, showing that secretomic analysis is suitable for the identification of protein biomarkers when combined with protein immunoassay. Using this method, we determined tenascin-X as a new potent marker for serous ovarian cancer.

HSA21 Single-Minded 2 (Sim2) Binding Sites Co-Localize with Super-Enhancers and Pioneer Transcription Factors in Pluripotent Mouse ES Cells.

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.

Biased allelic expression in human primary fibroblast single cells.

The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability.

Frequent cases of RAS-mutated Down syndrome acute lymphoblastic leukaemia lack JAK2 mutations.

Children with Down syndrome (DS) and acute lymphoblastic leukaemia (ALL) have poorer survival and more relapses than non-DS children with ALL, highlighting an urgent need for deeper mechanistic understanding of DS-ALL. Here, using full-exome or cancer genes-targeted sequencing of 42 ALL samples from 39 DS patients, we uncover driver mutations in RAS, (KRAS and NRAS) recurring to a similar extent (15/42) as JAK2 (12/42) mutations or P2RY8-CRLF2 fusions (14/42). RAS mutations are almost completely mutually exclusive with JAK2 mutations (P=0.016), driving a combined total of two-thirds of analysed cases. Clonal architecture analysis reveals that both RAS and JAK2 drove sub-clonal expansions primarily initiated by CRLF2 rearrangements, and/or mutations in chromatin remodellers and lymphocyte differentiation factors. Remarkably, in 2/3 relapsed cases, there is a switch from a primary JAK2- or PTPN11-mutated sub-clone to a RAS-mutated sub-clone in relapse. These results provide important new insights informing the patient stratification strategies for targeted therapeutic approaches for DS-ALL.

Role of prostate apoptosis response 4 in translocation of GRP78 from the endoplasmic reticulum to the cell surface of trophoblastic cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB) and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4), a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.

Expression of metalloproteinases 1, 2, 7, 9, and 12 in human cytotrophoblastic cells from normal and preeclamptic placentas.

OBJECTIVES: Preeclampsia is a specific pregnancy disorder which could be due, at least in part, to impaired invasion of trophoblastic cells. Since matrix metalloproteinases (MMPs) are the predominant proteases involved in trophoblastic invasion, we investigated and compared expression of MMP-1, 2, 7, 9 and 12 of cytotrophoblastic cells (CTB) purified from preeclamptic (PE) placentas to control CTB. MATERIAL AND METHODS: In order to evaluate invasive properties of cells, purified CTB were seeded on collagen-coated insert following boyden chamber principle and matrix metalloproteinases (MMPs) expression was evaluated by qPCR. RESULTS: Our results showed that PE CTB are less invasive than control CTB in vitro. In parallel, expression of MMPs, except for MMP-2, tends to be decreased in PE CTB compared to control CTB. CONCLUSION: At the exception of MMP-2, this study confirms the importance of MMPs in development of PE.