ABSTRACT
BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using the discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score < 4. Both cohorts were further separated into four groups according to molecular subtypes (TMB positive; MSI positive; HRD positive; all others). When grouped by molecular subtype, there was a significant difference between groups using an HRD ≥4 cutoff in the initial (p = 0.0024) and replication (p = 0.042) cohorts. The Hec1a model (HRD score = 19) was highly sensitive to olaparib in in vitro and in vivo experiments. CONCLUSIONS: High HRD score was associated with worse DFS in our patient cohort. These findings suggest that HRD score may have clinical utility in patients with advanced or recurrent endometrial cancer.
Subject(s)
Endometrial Neoplasms/genetics , Homologous Recombination/genetics , Female , Humans , Middle AgedABSTRACT
Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this process are currently available. Here, an integrative computational analysis of the Cancer Genome Atlas OC data set and experimental validation identifies a zinc finger transcription factor ZNF304 associated with OC metastasis. High tumoral ZNF304 expression is associated with poor overall survival in OC patients. Through reverse phase protein array analysis, we demonstrate that ZNF304 promotes multiple proto-oncogenic pathways important for cell survival, migration and invasion. ZNF304 transcriptionally regulates ß1 integrin, which subsequently regulates Src/focal adhesion kinase and paxillin and prevents anoikis. In vivo delivery of ZNF304 siRNA by a dual assembly nanoparticle leads to sustained gene silencing for 14 days, increased anoikis and reduced tumour growth in orthotopic mouse models of OC. Taken together, ZNF304 is a transcriptional regulator of ß1 integrin, promotes cancer cell survival and protects against anoikis in OC.
Subject(s)
Anoikis , Carcinoma/metabolism , Integrin beta Chains/metabolism , Ovarian Neoplasms/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Gene Silencing , HumansABSTRACT
The purpose of this study was to investigate the antitumor effects of a combination of metronomic doses of a novel delivery vehicle, PLGA-PRINT nanoparticles containing docetaxel, and antiangiogenic mEZH2 siRNA incorporated into chitosan nanoparticles. In vivo dose-finding studies and therapeutic experiments were conducted in well-established orthotopic mouse models of epithelial ovarian cancer. Antitumor effects were determined on the basis of reduction in mean tumor weight and number of metastatic tumor nodules in the animals. The tumor tissues from these in vivo studies were stained to evaluate the proliferation index (Ki67), apoptosis index (cleaved caspase 3), and microvessel density (CD31). The lowest dose of metronomic regimen (0.5 mg/kg) resulted in significant reduction in tumor growth. The combination of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA showed significant antitumor effects in the HeyA8 and SKOV3ip1 tumor models (P < 0.05). Individual as well as combination therapies showed significant antiangiogenic, antiproliferative, and proapoptotic effects, and combination therapy had additive effects. Metronomic delivery of PLGA-PRINT-docetaxel combined with CH-mEZH2 siRNA has significant antitumor activity in preclinical models of ovarian cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Nanoparticles/administration & dosage , Ovarian Neoplasms/drug therapy , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/genetics , RNA, Small Interfering/administration & dosage , Taxoids/administration & dosage , Administration, Metronomic , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Docetaxel , Enhancer of Zeste Homolog 2 Protein , Female , Gene Silencing/drug effects , Humans , Mice , Mice, Nude , Nanoparticles/chemistry , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Polycomb Repressive Complex 2/metabolism , RNA, Small Interfering/genetics , Randomized Controlled Trials as Topic , Taxoids/chemistryABSTRACT
Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of beta-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.
Subject(s)
Agrobacterium tumefaciens/enzymology , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Folding , beta-Glucosidase/chemistry , beta-Glucosidase/metabolism , Alcohols/pharmacology , Catalysis , Enzyme Stability/drug effects , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Plasmids/genetics , Protein Conformation/drug effects , Protein Denaturation/drug effects , Protein Renaturation/drug effects , Sodium Chloride/pharmacology , Substrate Specificity , Temperature , beta-Glucosidase/geneticsABSTRACT
A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.
