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.

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.

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.

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.